Flow Cytometry, Ektacytometry, DNA Ploidy, and S-phase Fraction

Number: 0351

Table Of Contents

Policy
Applicable CPT / HCPCS / ICD-10 Codes
Background
References

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Policy

Scope of Policy

This Clinical Policy Bulletin addresses flow cytometry, ektacytometry, DNA ploidy, and S-phase fraction.

1. Medical Necessity

   1. Flow Cytometry for Cell Surface Markers

      Aetna considers flow cytometry for cell surface markers medically necessary for any of the following conditions:

      1. Abnormal tissue, bone marrow, or blood histology when the results are suspicious for lymphoma, leukemia, or myelodysplastic syndrome and where the physician must distinguish reactive from neoplastic conditions; or
      2. B-cell monitoring for immunosuppressive disorders; or
      3. Hairy cell monitoring; or
      4. Hereditary persistence of fetal hemoglobin (HPFH), in persons with unexplained increases in hemoglobin F; or
      5. Hereditary spherocytosis, in persons with Coombs' negative hemolytic anemia; or
      6. Immunophenotyping for leukemia, lymphoma, or myelodysplastic syndrome; or
      7. Mast cell disease; or
      8. Measurement of CD4/CD8 ratio from bronchiolar lavage fluid for diagnosis of sarcoidosis; or
      9. Multiple myeloma; or
      10. Myeloproliferative neoplasms, for workup of disease progression to advanced phase or transformation to AML; or
      11. Paroxysmal nocturnal hemoglobinuria; or
      12. Post-operative monitoring of members who have undergone organ transplantation; or
      13. Primary immunodeficiencies; or
      14. Sezary syndrome, diagnosis; or
      15. T-cell monitoring for HIV infection and AIDS.

   2. Ploidy and Cell Proliferation Activity: Indications

      A National Institutes of Health consensus development conference concluded that measurement of flow cytometry-derived DNA content (ploidy), or cell proliferative activity (S-phase fraction or % S-phase) is not indicated for prognostic or therapeutic purposes in the routine clinical management of cancers.  Therefore, Aetna considers flow cytometry-derived DNA content (ploidy), or cell proliferative activity (S-phase fraction or % S-phase) in any of the following localized cancers without metastatic disease medically necessary only when the obtained prognostic information will affect treatment decisions:

      1. Endometrial adenocarcinoma; or
      2. Gastric cancer; or
      3. Mediastinal neuroblastoma; or
      4. Medulloblastoma; or
      5. Ovarian carcinoma; or
      6. Partial hydatidiform mole; or
      7. Prostatic adenocarcinoma; or
      8. Renal cell adenocarcinoma; or
      9. Urinary bladder carcinoma.

      Note: This test is usually performed only once per tumor lifetime usually after a diagnosis has been made and before treatment is initiated.

   3. Ektacytometry

      Aetna considers ektacytometry medically necessary for the diagnosis of red blood cell (RBC) cytoskeleton and hydration disorders (e.g., hereditary spherocytosis, pyro-poikilocytosis, stomatocytosis, ovalocytosis, elliptocytosis and xerocytosis) when RBC morphology does not provide a clear diagnosis.

2. Experimental, Investigational, or Unproven

   The following procedures are considered experimental, investigational, or unproven because the effectiveness of these approaches has not been established:

   1. Aetna considers measurement of an RBC adhesion index (e.g., Hypoxic BioChip Adhesion or Normoxic BioChip Adhesion) experimental, investigational, or unproven for all indications because the effectiveness of this approach has not been established.
   2. Aetna considers flow cytometry-derived DNA content (ploidy), or cell proliferative activity (S-phase fraction or % S-phase) experimental, investigational, or unproven in any of the following cancers (not an all-inclusive list) because its effectiveness for these indications has not been established:
      
      
      1. Breast cancer; or
      2. Cervical cancer; or
      3. Colorectal cancer; or
      4. Non-small cell lung cancer; or
      5. Pediatric intracranial tumors; or
      6. Small cell lung cancer.

   3. Aetna considers the following microfluidic flow adhesion assays/tests experimental, investigational, or unproven for sickle cell disease: (i) flow adhesion of whole blood to P-Selectin (WB-PSEL); and (ii) flow adhesion of whole blood on VCAM-1 (FAB-V) because their clinical values has not been established.

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Table:

CPT Codes / HCPCS Codes / ICD-10 Codes

Code	Code Description
CPT codes covered if selection criteria are met:
86360	T cells; absolute CD4 and CD8 count, including ratio
88182	Flow cytometry, cell cycle or DNA analysis
88184	Flow cytometry, cell surface, cytoplasmic, or nuclear marker, technical component only; first marker
+ 88185	each additional marker (List separately in addition to code for first marker)
88187	Flow cytometry, interpretation; 2 to 8 markers
88188	9 to 15 markers
88189	16 or more markers
CPT codes not covered for indications listed in the CPB:
0121U	Sickle cell disease, microfluidic flow adhesion (VCAM-1), whole blood
0122U	Sickle cell disease, microfluidic flow adhesion (P-Selectin), whole blood
0303U	Hematology, red blood cell (RBC) adhesion to endothelial/subendothelial adhesion molecules, functional assessment, whole blood, with algorithmic analysis and result reported as an RBC adhesion index; hypoxic
0304U	Hematology, red blood cell (RBC) adhesion to endothelial/subendothelial adhesion molecules, functional assessment, whole blood, with algorithmic analysis and result reported as an RBC adhesion index; normoxic
ICD-10 codes covered if selection criteria are met:
B20	Human immunodeficiency virus [HIV] disease [T cell monitoring]
C16.0 - C16.9	Malignant neoplasm of stomach [gastric, localized without metastatic disease]
C38.1 - C38.2	Malignant neoplasm of anterior and posterior mediastinum [neuroblastoma, localized without metastatic disease]
C54.1	Malignant neoplasm of endometrium [localized without metastatic disease]
C56.1 - C56.9	Malignant neoplasm of ovary [localized without metastatic disease]
C57.4	Malignant neoplasm of uterine adnexa, unspecified [localized without metastatic disease]
C61	Malignant neoplasm of prostate
C64.1 - C64.9	Malignant neoplasm of unspecified kidney, except renal pelvis [localized without metastatic disease]
C67.0 - C67.9	Malignant neoplasm of bladder [localized without metastatic disease]
C71.0 - C71.9	Malignant neoplasm of brain [medulloblastoma in adults only]
C81.00 - C86.61 C88.40 - B88.41, C88.80 - C88.91 C90.00 - C94.42 C94.80 - C95.91 C96.0 - C96.4 C96.A - C96.9	Malignant neoplasm of lymphoid, hematopoietic and related tissue
D00.2	Carcinoma in situ of stomach [gastric]
D07.0	Carcinoma in situ of endometrium
D07.5	Carcinoma in situ of prostate
D09.0	Carcinoma in situ of bladder
D43.0 - D43.3	Neoplasm of uncertain behavior of brain and cranial nerves [intracranial in adults only]
D45	Polycythemia vera
D46.0 - D46.9	Myelodysplastic syndromes
D47.01 - D47.1 D47.3 - D47.9	Other neoplasms of uncertain behavior of lymphoid, hematopoietic and related tissue
D49.6	Neoplasm of unspecified behavior of brain [adults only]
D56.4	Hereditary persistence of fetal hemoglobin [HPFH]
D57.00 - D57.3 D57.80 - D57.819	Sickle-cell disorders
D58.0	Hereditary spherocytosis
D58.2	Other hemoglobinopathies
D59.5 - D59.8	Acquired hemolytic anemia
D75.81	Myelofibrosis
D80.0 - D81.2, D81.4 D81.89 - D82.1 D83.0 - D84.9 D89.810 - D89.9	Certain disorders involving the immune mechanism [B-cell monitoring for immunosuppressive disorders and primary immunodeficiencies]
D86.0 - D86.9	Sarcoidosis
O01.0 - O01.9	Hydatidiform mole
R89.7	Abnormal histological findings in specimens from other organs, systems and tissues
T86.00 - T86.99	Complications of transplanted organs and tissue [postoperative monitoring]
Z21	Asymptomatic human immunodeficiency virus [HIV] infection status [T cell monitoring]
Z94.0 - Z94.9, Z95.3	Transplanted organ and tissue status [postoperative monitoring]
ICD-10 code not covered for indications listed in the CPB:
C18.0 - C21.8	Malignant neoplasm of colon, rectosigmoid junction, rectum, anus and anal canal
C34.00 - C34.92	Malignant neoplasm of bronchus and lung [non-small cell lung cancer]
C50.011 - C50.929	Malignant neoplasm of breast
C53.0 - C53.9	Malignant neoplasm of cervix uteri
C71.0 - C71.9	Malignant neoplasm of brain [pediatric only]
D43.0 - D43.3	Neoplasm of uncertain behavior of brain and cranial nerves [pediatric intracranial only]
D49.6	Neoplasm of unspecified behavior of brain [pediatric only]
Ektacytometry:
CPT codes covered if selection criteria are met:
0305U	Hematology, redbloodcell (RBC) functionality and deformity as a function of shear stress, whole blood, reported as a maximum elongation index
ICD-10 codes covered if selection criteria are met:
D58.0	Hereditary spherocytosis
D58.1	Hereditary elliptocytosis [Ovalocytosis]
D58.8	Other specified hereditary hemolytic anemias [Stomatocytosis, Xerocytosis]
R71.8	Other abnormality of red blood cells [pyro-poikilocytosis]

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Background

Flow cytometry is an emerging technique, which involves the separation, classification and quantitation of cell types by:

1. cell surface antigens (phenotype);
2. DNA content (ploidy) (DNA index); and
3. DNA flow cytometric proliferation analysis (S-phase fraction or % S-phase).

The basic technique involves passage of a monocellular stream of cells through a beam of laser light after cell surface antigens have been tagged with fluorescent monoclonal antibodies; complex computerized instruments are then used to sort normal from abnormal cells and also subgroups of the same cell type.  Data are most often collected as a bar-graph histogram, which is then displayed visually as a densitometer tracing of the bar graph; the concentration of cells in each bar appears as a separate peak for each cell category, with a peak height proportional to the number of cells in each bar of the bar graph.  For example, lymphocytes can be separated into B- and T-cell categories; the T-cells can be further phenotyped as helper/inducer, suppressor/cytotoxic, or natural killer cell types.

Using fluorescent dye that stains nucleic acids, flow cytometry methods have also been applied to measure nuclear deoxyribonucleic acid (DNA) content (ploidy) as a prognostic indicator of solid tumors based on the fact that malignant cells sometimes show abnormalities in total chromosome number and the frequency of these abnormalities generally increases with progression to higher-grade tumors.  In such testing, DNA diploid tumors are those in which a single peak containing an amount of DNA similar to normal control cells is generated by flow cytometry.  DNA aneuploid tumors have additional peaks on DNA histogram, presumably representing cells containing more or less nucleic acid than is found in 46 normal chromosomes.  A more quantitative method of expression is the DNA index (DI), which is the ratio of the mean tumor sample G0 /G1 DNA content of normal diploid reference cells.  The greater the deviation of the DI from 1, the more "aneuploid" the tumor.

Another type of measurement for DNA is the assessment of % S-phase or the S-phase fraction (SPF), the percentage or proportion of cells preparing for mitosis by their active doubling of DNA.  Since tumor cells tend to replicate more readily than normal cells, increased SPF activity can therefore raise the question of malignancy.  In many tumors, a high SPF correlates positively with poor differentiation, increasing tumor size, and degree of aggressiveness in tumor spread, which all have prognostic significance.

However, controversy exists concerning the use of DNA content (aneuploid versus diploid status) as an independent prognostic indicator.  Basic and clinical studies have reached different conclusions concerning its value.  While many of the earlier studies reported that diploid carcinomas had significantly or considerably better prognosis than aneuploid ones, some more recent studies do not confirm this or do not find that ploidy is a significant independent risk factor.  The results of studies on DNA content in different types of tumors have yielded varying results.  Aneuploidy, which is thought to be most often associated with malignancy, has been shown to occur in some non-neoplastic cell populations as part of the reaction to or regeneration after inflammation or tissue destruction and has also been reported in some benign tumors.  While groups of diploid patients have better prognoses than groups of non-diploid patients, ploidy status may have uncertain prognostic value in individual patients.  A small biopsy demonstrating diploid tumor may be missing a significant underlying non-diploid component.  Controversy remains as to the nature of the relationship between histologic grade and tumor stage with the degree of aneuploidy.  And, lastly, standards for tissue preparation and analysis to insure reproducibility are not yet established from lab to lab.

Increased S-phase activity is somewhat better accepted as an unfavorable prognostic sign.  Unfortunately, SPF is technically more difficult to measure accurately and its usefulness is limited by the lack of mutually accepted technical standards and methodologies between laboratories, including a uniform procedure for assaying cancers with multiple subpopulations of different kinetics.  Not all tumors with increased S-phase fraction are malignant; not all malignant tumors with increased S-phase fraction metastasize; and not all malignant tumors with relatively small S-phase fraction fail to metastasize.  Moreover, the S-phase peak is usually not large, even when considerable S-activity is occurring.  In DNA aneuploid tumors, the multiple peaks each represent a different cellular population; however, proliferation results are generally reported from only one peak, complicating the interpretation of the results.  Another problem of definition is that of high versus low S phase; these are frequently defined retrospectively or arbitrarily as being above or below the median for all samples assayed.  Such post hoc definitions may introduce bias and should in the future be replaced by laboratory-specific, prospectively determined, biologically relevant cutoffs.

The American Society of Clinical Oncology (2001) prepared evidence-based guidelines on the use of tumor markers in breast cancer and colorectal cancer, and reached the following conclusions.  Regarding DNA ploidy or flow cytometric proliferation analysis as a marker for colorectal cancer, ASCO concluded that "[p]resent data are insufficient to recommend DNA flow cytometrically-derived ploidy (DNA index) for the management of colorectal cancer."  Regarding the use of DNA flow cytometrically derived parameters as markers for breast cancer, ASCO concluded that:

1. "[p]resent data are insufficient to recommend obtaining DNA flow cytometry-derived estimates of DNA content or S-phase in breast tissue"; and
2. "DNA flow cytometry-derived ploidy are not recommended to be used to assign a patient to prognostic groupings.   There is insufficient evidence to recommend the use of S-phase determination for assigning patients to prognostic groupings."

In conclusion, DNA flow cytometry-derived estimates of DNA ploidy and S-phase proliferation have been correlated with cancer patient outcome in many studies.  However, the evidence is almost entirely from retrospective studies with multiple cut-off points for defining high- and low-risk populations.  The final value and role for DNA ploidy and S-phase fraction testing in current clinical practice remains to be finally determined.  The data are contradictory (hence the present controversy between the NIH and CMS's coverage policies) as to whether the prognostic information is independent of other recognized predictors, particularly stage, and further evaluation is required to determine if these techniques will provide useful prognostic information regarding individual patient survival, health outcome, and response to therapy.

The NCCN Biomarkers Compendium (2017) recognizes the use of flow cytometry for the following indications:

• Acute lymphoblastic leukemia

  • Comprehensive flow cytometric immunophenotyping to include B, T and myeloid lineage markers;
  • Year 1 (every 1-2 months), bone marrow aspirate as indicated. If bone marrow aspirate is done: Flow cytometry with additional studies;
  • Minimal residual disease assessment - The most frequently employed methods for MRD assessment includes multicolor flow cytometry to detect abnormal immuno-phenotypes;
    
    
• Acute myeloid leukemia: flow cytometry and IHC for evaluation of acute leukemia;
• Chronic lymphocytic leukemia / small lymphocytic lymphoma: determination of CD38 and Zap 70 expression by flow cytometry, methylation or immunohistochemistry informative for prognostic or therapy determination;
• Chronic myelogenous leukemia: flow cytometry as additional testing to determine cell lineage in advanced phase (accelerated phase or blast phase);
• Multiple myeloma
  
  
  • Initial diagnostic workup: Unilateral bone marrow aspirate + biopsy, including bone marrow immunohistochemistry and/or bone marrow flow cytometry;
  • Smoldering (asymptomatic) clinical presentation - Multiparameter flow cytometry as clinically indicated for follow up/surveillance;
    
    

• Myelodysplastic syndromes

  • Additional testing: Consider flow cytometry (FCM) to evaluate for PNH clone;
  • Helpful in some clinical situations:
    
    
    • Consider flow cytometry (FCM) for MDS diagnostic aid;
    • Consider flow cytometry (FCM) to assess possible large granular lymphocytic (LGL) disease;
      
      
• Myeloproliferative neoplasms: advanced phase/AML workup;
• Non-Hodgkin lymphoma: diagnosis.

Habermehl et al (2004) examined flow-cytometric DNA values of pediatric intra-cranial tumors and attempted to establish DNA analysis as a potential prognostic parameter.  A total of 29 brain tumor specimens from 26 pediatric patients were cryo-preserved within a 3-year period.  The DNA content was measured by flow cytometry.  Six of the tumor specimens had aneuploid DNA patterns.  The median of the proliferation index was lower in the survivor group compared with the non-survivor group (36.4 and 47.5 %, respectively).  Ten of the 26 patients are still alive, 8 were lost to follow-up, and 8 died.  Flow-cytometric DNA analysis may be a helpful tool for examining brain tumors in children.  The small size of this study could not establish flow cytometry as a definite prognostic factor.  Further prospective multi-center studies are needed to assess the prognostic significance of flow-cytometric DNA analysis.

Gastric cancer is still a common cause of cancer-related deaths worldwide, despite improved diagnostic and therapeutic implications.  Hence, early diagnosis has critical importance.  Flow cytometry reveals rapid and reproducible quantification of nuclear DNA content of disaggregated tissues and assessment of its significance in various malignant and pre-cancerous lesions.  In a multi-center study, Yasa and colleagues (2005) examined 121 patients with gastric cancer, chronic atrophic gastritis, gastric polyps, intestinal metaplasia, and gastric dysplasia as well as 36 healthy controls.  Flow cytometric measurements of DNA ploidy, total S-phase, G2M-phase and proliferative indexes were analyzed on fresh gastric biopsy specimens obtained by gastroscopy.  DNA aneuploidy was present in 43.8 % of the gastric cancers (p < 0.05).  These investigators found a DNA aneuploidy rate of 15.4 % in chronic atrophic gastritis, 15.4 % in intestinal metaplasia and 25 % in epithelial dysplasia.  One of 9 polyps had aneuploidy.  None of the normal gastric mucosa samples showed aneuploidy.  The controls had lower rates of total S-phase and proliferative indexes (p < 0.05).  The authors concluded that DNA flow cytometry may be offered as an objective diagnostic tool for early detection of malignant transformation in gastric lesions.  Moreover, Russo et al (2001) who stated that DNA ploidy and SPF, when associated with clinico-pathological staging, might be useful for the identification of gastric cancer patients who have different risks for death or relapse of disease.  In addition, Jiao et al (2004) noted that SPF may be a more useful indicator of aggressive behavior in gastric cancers than DNA aneuploidy.

The American Society of Clinical Oncology's update of recommendations for the use of tumor markers in breast cancer (Harris et al, 2007) noted that there is insufficient evidence to support routine use in clinical practice of DNA/ploidy by flow cytometry.  ASCO's updated recommendations on the use of tumor markers in colorectal cancer state that neither flow-cytometrically derived DNA ploidy nor DNA flow cytometric proliferation analysis (% S phase) should be used to determine prognosis of early-stage colorectal cancer (Locker et al, 2006).

Wolfson et al (2008) examined possible associations between measurements of DI, SPF, and tumor heterogeneity (TH) using flow cytometry and overall survival (OS) for patients with invasive cervical carcinoma treated with definitive irradiation.  A total of 57 patients with International Federation of Obstetrics and Gynecology Stages IB(2) through IVB cervical carcinomas treated with definitive radiotherapy with or without concurrent chemotherapy were enrolled into this registry study that involved flow cytometric analysis of fresh tissue from each cervical cancer obtained by pre-treatment biopsy.  These specimens were evaluated for DNA aneuploidy (DI less than or equal to 1.5 versus greater than 1.5), SPF (less than or equal to 15 % versus greater than 15 %), and TH (uniploid versus multiploid).  In these analyses, 27 of the patients were treated in Radiation Therapy Oncology Group protocol 9001, and an additional 30 were offered chemoradiation at a single institution.  Forty-one patients had DI less than or equal to 1.5 and 16 DI greater than 1.5.  Twenty-nine patients had SPF less than or equal to 15 %, 26 greater than 15 %, and 2 had no determinable SPF.  Forty-three patients had uniploid and 14 multiploid tumors.  The 4-year estimated OS rate for the entire study cohort was 62 % (95 % confidence interval: 48 % to 74 %).  With a median follow-up of 3.7 years, there were no observable associations by univariate analysis for DI, SPF, or TH concerning patient survival.  The authors concluded that there were no statistically significant associations among DI, SPF, or TH and patient outcome.  They stated that additional studies are needed to identify tumor biomarkers that could predict patients at risk for disseminated disease.  Furthermore, Dabic et al (2008) stated that clinical stage and architectural grade are significant predictors for survival of patients with cervical adenocarcinoma.  Status of HPV infection, flow cytometric parameters, nuclear grade and menstrual status do not predict patient survival.

Suehiro and colleagues (2008) stated that many investigators have reported that aneuploidy detected by flow cytometry is a useful prognostic marker in patients with endometrial cancer.  Laser scanning cytometry (LSC) is a technology similar to flow cytometry but is more feasible for clinical laboratory use.  These investigators evaluated the usefulness of DNA ploidy detected by LSC as a prognostic marker in patients with endometrial cancer and examined genetic and epigenetic factors related to aneuploidy.  Endometrial cancer specimens from 106 patients were evaluated.  The methylation status of CDH13, Rassf1, SFRP1, SFRP2, SFRP4, SFRP5, p16, hMLH1, MGMT, APC, ATM, and WIF1 and mutations in the p53 and CDC4 genes were investigated.  Laser scanning cytometry was performed to determine DNA ploidy.  Fluorescence in situ hybridization was done with chromosome-specific centromeric probes to assess chromosomal instability.  Uni-variate and multi-variate analyses revealed that p53 mutation and lack of CDH13 hypermethylation associated positively with aneuploidy.  Uni-variate analysis showed that aneuploidy, chromosomal instability, and lack of CDH13 hypermethylation as well as surgical stage were significantly predictive of death from endometrial cancer.  Furthermore, multi-variate analysis revealed that stage in combination with either DNA aneuploidy or lack of CDH13 hypermethylation was an independent prognostic factor.  The authors concluded that these findings suggested that analysis of DNA ploidy and methylation status of CDH13 may help predict clinical outcome in patients with endometrial cancer.  Moreover, they stated that prospective randomized trials are needed to confirm the validity of an individualized approach, including determination of tumor ploidy and methylation status of CDH13, to management of endometrial cancer patients.

Ludovini et al (2008) assessed the relationship between a panel of biological markers (p53, Bcl-2, HER-2, Ki67, DNA ploidy and S-phase fraction) and clinical-pathological parameters and its impact on outcome in non-small cell lung cancer (NSCLC).  Tumor tissue specimens obtained following surgical resection were collected from consecutive patients with NSCLC.  These researchers used an immunocytochemical technique for p53, Bcl-2, HER-2 and Ki67 analysis in fine-needle aspirates obtained from surgical samples that were also evaluated by flow cytometric DNA analysis using a FACScan flow cytometer.  From April 2000 to December 2005, a total of 136 patients with radically resected NSCLC were recruited.  Median age was 66 years (range of 31 to 84 years), male/female ratio 117/19, ECOG performance status 0/1 127/4, stage I/II/III 76/25/35, squamous/adenocarcinoma/large-cell/mixed histology 62/49/17/8, smokers yes/no 121/11.  Positivity of p53, Bcl-2, HER-2 and Ki67 was detected in 51.4 %, 27.9 %, 25.0 % and 55.8 % of the samples, respectively; 82.9 % of the cases revealed aneuploid DNA histograms and 56.7 % presented an S-phase fraction of more than 12 %.  Statistically significant associations between high Ki67 and poorly differentiated tumors (p = 0.016) and a smoking history (p = 0.053); p53 positivity and high Ki67 (p = 0.002); HER-2 positivity and adenocarcinoma subtype (p = 0.015) and presence of lymph node involvement (p = 0.006); and Bcl-2 positivity and squamous cell carcinoma subtype (p = 0.058) were observed.  At uni-variate analysis, high Ki67 proved to be the only marker associated with disease-free survival (p = 0.047).  After adjusting for stage, none of the examined immunocytochemical markers emerged as an independent factor for disease-free and overall survival; only pathological stage was identified as an independent prognostic factor for disease-free survival (p = 0.0001) and overall survival (p = 0.0001).  In the group of 76 patients classified as TNM stage I, high Ki67 was the only marker associated with recurrence of disease (p = 0.05).  The authors concluded that these findings do not support a relevant prognostic role of immunocytochemical markers in NSCLC, even if the Ki67 index might have particular relevance to identify patients with more aggressive tumors who are at high risk for disease relapse.

The American Thoracic Society’s "Statement on Sarcoidosis" (1999) stated that "In some instances, bronchoalveolar lavage (BAL) and studies on lymphocyte subpopulations are helpful.  According to Costabel, a CD41CD8 ratio greater than 3.5 has a sensitivity of 53 %, a specificity of 94 %, a positive-predictive value of 76 % and a negative-predictive value of 85 %.  In other words, a CD4/CD8 ratio greater than 3.5 provides a diagnosis of sarcoidosis with a specificity of 94 % even if the TLB has not been diagnostic".

Furthermore, an UpToDate review on "Clinical manifestations and diagnosis of pulmonary sarcoidosis" (King, 2015) states that "Bronchoalveolar lavage -- BAL can be used as an adjunctive measure to support the diagnosis of sarcoidosis by demonstrating a reduced number of CD8 cells, an elevated CD4 to CD8 ratio, and an increased amount of activated T cells, CD4 cells, immunoglobulins, and IgG-secreting cells.  BAL is also used to exclude infections and malignancy as alternative diagnoses".

National Comprehensive Cancer Network's guidelines on myeloproliferative neoplasms (NCCN, 2017) states that bone marrow aspirate and biopsy with reticulin stain and bone marrow cytogenetics, flow cytometry, and molecular testing for AML-associated mutations is recommended as part of initial workup of disease progression to advanced stage or transformation to AML.

Small Cell Lung Cancer

An UpToDate review on "Overview of the initial evaluation, treatment and prognosis of lung cancer" (Midthun, 2016) does not mention flow cytometry as a management tool.

Furthermore, National Comprehensive Cancer Network’ clinical practice guideline on "Small cell lung cancer" (Version 1.2016) does not mention flow cytometry as a management tool.

Osmotic Gradient Ektacytometry

Osmotic gradient ektacytometry (OGE; osmoscan) is part of the laboratory diagnosis process of hereditary spherocytosis (HS) and other red blood cell (RBC) membrane disorders.

Parrow and associates (2018) noted that decreased RBC deformability is characteristic of several disorders.  In some cases, the extent of defective deformability could predict severity of disease or occurrence of serious complications.  Ektacytometry uses laser diffraction viscometry to measure the deformability of RBCs subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress.  However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid as well as deformable RBCs.  This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability.  Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood.  In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin (Hb) concentration and Hb composition of RBCs.  In addition to measuring deformability, OGE provides information regarding the osmotic fragility and hydration status of RBCs.  These parameters also reflect the Hb composition of RBCs from sickle cell patients.  Ektacytometry measures deformability in populations of RBCs and does not, therefore, provide information on the deformability or mechanical properties of individual RBCs.  The authors concluded that these techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders; and sickle cell anemia is one well-characterized example.  Other potential disorders where measurements of RBC deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and hemolytic anemias due to membrane defects.

Llaudet-Planas and colleagues (2018) stated that new generation OGE has become a powerful procedure for measuring RBC deformability and thus for the diagnosis of RBC membrane disorders.  These investigators provided further support to the usefulness of OGE for the differential diagnosis of HS by measuring the optimal cut-off values of the parameters provided by this technique.  A total of 65 cases of HS, 7 hereditary elliptocytosis (HE), 3 hereditary xerocytosis (HX), and 171 normal controls were analyzed with OGE in addition to the routine RBC laboratory techniques.  The most robust OGE parameters for HS diagnosis were determined using receiver operating characteristic (ROC) curve analysis.  The best diagnostic criteria for HS were the combination of decreased minimal elongation index up to 3 % and increased minimal osmolality point up to 5.2 % when compared to the mean of controls.  Using this established criterion, OGE reported a sensitivity of 93.85 % and a specificity of 98.38 % for the diagnosis of HS.  The authors concluded that OGE is an effective diagnostic test for HS and enables its differential diagnosis with other RBC membrane diseases based on specific pathology profiles.

Huisjes and associates (2020) noted that HS originates from defective anchoring of the cytoskeletal network to the trans-membrane protein complexes of RBCs.  Red cells in HS are characterized by membrane instability and reduced deformability and there is marked heterogeneity in disease severity among patients.  To unravel this variability in the severity of HS, these researchers analyzed blood samples from 21 HS patients with defects in ankyrin, band 3, α-spectrin or β-spectrin by means of red cell indices, eosin-5-maleimide binding, microscopy, the osmotic fragility test, Percoll density gradients, vesiculation and OGE to evaluate cell membrane stability, cellular density and deformability.  Reticulocyte counts, CD71 abundance, band 4.1 a:b ratio, and glycated Hb were used as markers of RBC turnover.  These investigators observed that patients with moderate/severe HS have short-living RBCs of low density and abnormally high intercellular heterogeneity.  These cells showed a prominent decrease in membrane stability and deformability and, as a consequence, were quickly removed from the circulation by the spleen.  In contrast, in mild HS less pronounced reduction in deformability resulted in prolonged RBC lifespan and, hence, cells were subjected to progressive loss of membrane.  RBCs from patients with mild HS thus become denser before they were taken up by the spleen.  The authors concluded that RBC membrane loss, cellular heterogeneity and density were strong markers of clinical severity in patients with HS.

Zaidi et al (2020) stated that the measurement of band 3 (AE1, SLC4A1, CD233) content of RBCs by eosin-5- maleimide (EMA) staining is swiftly replacing conventional osmotic fragility (OF) test as a tool for laboratory confirmation of HS world-while.  These researchers had systematically evaluated the EMA test as a method to screen for a variety of anemias in the past decade; and compared these findings to those obtained with the OGE (osmoscan), which they had used in the last 30 years.  Their overall experience allowed them to characterize the distinctive patterns with the 2 tests in several congenital erythrocyte membrane disorders, such as HS, HE, Southeast Asian ovalocytosis (SAO), hereditary pyro-poikilocytosis (HPP) variants, erythrocyte volume disorders, various red cell enzymopathies, and hemoglobinopathies.  A crucial difference between the 2 methodologies is that OGE measures RBC deformability of the entire sample of RBCs, while the EMA test examines the band 3 content of individual RBCs.  EMA content is influenced by cell size as smaller RBCs have lower amount of total membrane than larger cells.  The SAO mutation alters the EMA binding site resulting in a lower EMA MCF even as the band 3 content itself is unchanged.  The authors conclude that EMA scan results should be interpreted with caution and both the histograms and dot plots should be analyzed in the context of the clinical picture and morphology.

Berrevoets et al (2021) stated that HS is the most common form of hereditary chronic hemolytic anemia.  It is caused by mutations in RBC membrane and cytoskeletal proteins, which compromise membrane integrity, leading to vesiculation.  This would result in entrapment of poorly deformable spherocytes in the spleen.  Splenectomy is a procedure often carried out in patients with HS. The clinical benefit results from removing the primary site of destruction; thus, improving RBC survival.  However, whether changes in RBC properties contribute to the clinical benefit of splenectomy is unknown.  These researchers employed ektacytometry to examine the longitudinal effects of splenectomy on RBC properties in 5 well-characterized HS patients at 4 different time-points and in a case-control cohort of 26 HS patients; OGE showed that splenectomy resulted in improved intra-cellular viscosity (hydration state) whereas total surface area (TSA) and surface-to-volume ratio remained essentially unchanged.  The cell membrane stability test (CMST), which evaluates the in-vitro response to shear stress, showed that after splenectomy, HS RBCs had partly regained the ability to shed membrane, a property of healthy RBCs, which was confirmed in the case-control cohort.  In particular the CMST holds promise as a novel biomarker in HS that reflects RBC membrane health and may be used to examine treatment response in HS.

Furthermore, an UpToDate review on “Hereditary elliptocytosis and related disorders” (Mentzer, 2021) states that “Osmotic gradient ektacytometry (OGE) measures the deformability and hydration of an RBC population.  OGE is effective in confirming the diagnosis when RBC morphology does not provide a clear diagnosis.  OGE is also helpful in distinguishing HE from hereditary spherocytosis (HS) and hereditary xerocytosis.  OGE can also measure the mechanical stability of RBC ghosts, which has been shown to be abnormal in individuals with HPP”.

RBC Adhesion Index

Dupuy and associates (2019) stated that micro-fluidic devices have become an integral method of cardiovascular research as they enable the study of shear force in biological processes, such as platelet function and thrombus formation.  In addition, micro-fluidic chips offer the benefits of ex-vivo testing of platelet adhesion using small amounts of blood or purified platelets.  Micro-fluidic chips comprise flow channels of varying dimensions and geometries that are connected to a syringe pump.  The pump draws blood or platelet suspensions through the channel(s) allowing for imaging of platelet adhesion and thrombus formation by fluorescence microscopy.  The chips can be fabricated from various blood-compatible materials.  The current protocol uses commercial plastic or in-house polydimethylsiloxane (PDMS) chips.  Commercial biochips offer the advantage of standardization whereas in-house chips offer the advantage of decreased cost and flexibility in design.  The authors concluded that micro-fluidic devices are a powerful tool to study the biorheology of platelets and other cell types with the potential of a diagnostic and monitoring tool for cardiovascular diseases.

Man and colleagues (2020) noted that up-regulated expression of P-selectin on activated endothelium and platelets significantly contributes to the initiation and progression of vaso-occlusive crises (VOC), a major cause of morbidity in sickle cell disease (SCD).  Crizanlizumab (Adakveo), a humanized monoclonal antibody against P-selectin, primarily inhibits the interaction between leukocytes and P-selectin; and has been shown to decrease the frequency of VOCs in clinical trials.  However, the lack of reliable in-vitro assays that objectively measure leukocyte adhesion to P-selectin remains a critical barrier to assessing and improving the therapeutic treatment in SCD.  These researchers presented a standardized micro-fluidic BioChip whole blood adhesion assay to evaluate leukocyte adhesion to P-selectin under physiologic flow conditions.  The findings demonstrated heterogeneous adhesion by leukocytes to immobilized P-selectin, and dose-dependent inhibition of this adhesion following pre-exposure to crizanlizumab.  More importantly, treatment with crizanlizumab following adhesion to P-selectin promoted detachment of rolling, but not of firmly adherent leukocytes.  The authors concluded that these promising findings suggested that the in-vitro microfluidic whole blood adhesion assay, the SCD Biochip, has the potential to enhance the understanding of the underlying mechanisms needed to develop targeted therapeutic strategies during drug development and in clinical trials.  These researchers are currently examining if these assays can predict individual patient response to targeted therapies.

Xuan et al (2018) noted that the immunoassays that are available for the serological diagnosis of the more common subtypes of auto-immune blistering diseases (AIBD) such as pemphigus vulgaris (PV) and pemphigus foliaceus (PF) include enzyme-linked immunosorbent assay (ELISA) testing to specific antigens desmoglein (Dsg)1 and Dsg3, direct immunofluorescence (DIF), indirect immunofluorescence (IIF), and immune-blotting.  These investigators carried out a review of the literature on the BIOCHIP assay.  A total of 6 studies examined the validity of a new BIOCHIP, mosaic-based, IIF test in patients with pemphigus and demonstrated its relatively high sensitivity and specificity (Dsg3: 97.62 to 100 %, 99.6 to 100 %; Dsg1: 90 %, 100 %) in comparison with ELISA (Dsg3: 81 to 100 %, 94 to 100 %; Dsg1: 69 to 100 %, 61.1 to 100 %), and/or IIF (PV: 75 to 100 %, 91.8 to 100 %; PF: 67 to 100 %) using suitable substrates.  So far, validation studies of the BIOCHIP have been carried out in 4 countries (Germany, Italy, Poland, and Turkey) but none in the southern hemisphere.  Caucasian patients were recruited as normal controls for these studies; therefore, the diagnostic value of the BIOCHIP remains uncertain in population groups of other ethnicities.  A range of disease control patients were recruited including patients with linear immunoglobulin A dermatosis, psoriasis, discoid lupus erythematosus, lichen planus, and non-inflammatory skin diseases (e.g., squamous cell carcinoma, basal cell carcinoma, and vascular leg ulcers).  The authors concluded that prospective studies with control patients from a diverse range of ethnicities are needed to better validate the BIOCHIP.  Moreover, these researchers noted that drawbacks of the studies identified in this review included small sample sizes, low case-to-control ratios, and selection bias; they stated that further investigations are needed to address these issues and to validate the use of the BIOCHIP in the diagnosis of pemphigus.

Arunprasath and co-workers (2020) stated that AIBD are a heterogeneous group of diseases characterized by auto-antibodies against desmosomal proteins in the pemphigus group of disorders and adhesion molecules of the dermal-epidermal junction in pemphigoid group of diseases.  DIF establishes the diagnosis of AIBD by demonstrating intercellular deposits of IgG and C3 in case of pemphigus and linear deposits of IgG and C3 along the basement membrane zone (BMZ) in bullous pemphigoid (BP).  BIOCHIP mosaic-based IIF, a novel diagnostic approach employs detection of characteristic staining pattern and target antigens in a single miniature incubation field.  In a cross-sectional study, these researchers compared the BIOCHIP mosaic-based IIF with DIF in the diagnosis of AIBD.  A total of 40 patients of AIBD in the active phase of the disease were included in the study.  Skin biopsy was carried out in these patients for DIF study and serum was subjected to BIOCHIP mosaic-based IIF assay.  The results were then compared.  DIF revealed a diagnosis of pemphigus in 18 patients and BP in 22 patients.  BIOCHIP showed a diagnosis of pemphigus in 18 patients, BP in 18 patients and floor pattern staining in 4 patients, which could be attributed to any of the floor pattern staining sub-epidermal blistering disease.  The authors concluded that the findings of this study showed that the diagnosis of AIBD by BIOCHIP is showing a statistically significant correlation with that of DIF.  Moreover, these researchers stated that drawbacks of this study included small sample size (n = 40), the lack of a control group and no comparison made with ELISA.

Nili et al (2021) noted that PV is an intra-epidermal AIBD characterized by auto-antibodies against desmosomal adhesion proteins, most commonly Dsg3, leading to the supra-basal cleft formation and acantholysis.  DIF and IIF studies display the intercellular deposition of IgG/C3 throughout the epidermis and presence of circulating auto-antibodies, respectively, as a net-like pattern.  However, the target antigen remains unknown using immune-fluorescence techniques.  Thanks to the development of Dsg ELISA, using recombinant technology, circulating antibodies against Dsg1 and 3 could be detected sensitively.  It is possible to differentiate PV from PF by means of this assay.  BIOCHIP mosaic and multi-variant ELISA are 2 novel serologic methods with the added value of the ability to screen several AIBDs simultaneously.  Non-Dsg1/3 antigens are also involved in the pathogenesis of PV and investigated more deeply thanks to the protein microarrays technique.  furthermore, patients with high values of anti-Dsg1/3 may be lesion-free, suggesting the presence of non-pathogenic auto-antibodies.  The authors concluded that newer diagnostic methods to replace traditional techniques should possess high sensitivity and specificity and be widely available, non-invasive, and relatively cheap.  Moreover, these researchers stated that these newly developed methods need to be further investigated before being recommended for routine use.

Microfluidic Flow Adhesion Assays/Tests for Sickle Cell Disease

Man et al (2020) noted that up-regulated expression of P-selectin on activated endothelium and platelets significantly contributes to the initiation and progression of VOC, a major cause of morbidity in sickle cell disease (SCD).  Crizanlizumab (ADAKVEO), a humanized monoclonal antibody against P-selectin, primarily inhibits the interaction between leukocytes and P-selectin, and has been reported to lower the frequency of VOCs in clinical trials.  However, the lack of reliable in-vitro assays that objectively measure leukocyte adhesion to P-selectin remains a critical barrier to assessing and improving the treatment of patients with SCD.  These investigators presented a standardized micro-fluidic BioChip whole blood (WB) adhesion assay to examine leukocyte adhesion to P-selectin under physiologic flow conditions.  The results showed heterogeneous adhesion by leukocytes to immobilized P-selectin, and dose-dependent inhibition of this adhesion following pre-exposure to crizanlizumab.  More importantly, treatment with crizanlizumab following adhesion to P-selectin promoted detachment of rolling, but not of firmly adherent leukocytes.  The authors concluded that the findings of this study suggested that the in-vitro micro-fluidic WB adhesion assay, the SCD Biochip, has the potential to enhance the understanding of the underlying mechanisms needed to develop targeted therapeutic strategies during drug development and in clinical trials.  Moreover, these researchers were examining if these assays could also predict individual patient response to targeted therapies.

Morikis et al (2021) stated that while neutrophils are essential in protecting the host against invading pathogens, they could also promote disease progression in SCD by becoming adherent to inflamed microvascular networks in peripheral tissue throughout the body.  During the inflammatory response, leukocytes extravasate from the blood-stream using selectin adhesion molecules and migrate to sites of tissue insult via activation of integrins that are essential for combating pathogens.  However, during VOC associated with SCD, neutrophils are activated during tethering and rolling on selectins up-regulated on activated endothelium that line blood vessels.  Recently, these researchers reported that recognition of sLex on L-selectin by E-selectin during neutrophil rolling initiated shear force resistant catch-bonds that facilitated tethering to endothelium and activation of integrin bond clusters that anchored cells to the vessel wall.  Evidence indicated that blocking this important signaling cascade would prevent the congestion and ischemia in micro-vasculature that occurred from neutrophil capture of sickled red blood cells, which are normally deformable ellipses that flow easily through small blood vessels.  Two recently completed clinical trials of therapies targeting selectins and their effect on neutrophil activation in small blood vessels demonstrated the importance of mechano-regulation that in health is an immune adaption facilitating rapid and proportional leukocyte adhesion, while sustaining tissue perfusion.  These investigators noted that while E-selectin and P-selectin mediate the capture of individual neutrophils, it is important to highlight heterotypic adhesive interactions in the genesis of VOC.  Secondary capture of free-flowing neutrophils, platelets and erythrocytes with adherent WBC are implicated in various disease states such as atherosclerotic plaque formation and VOC.  Homotypic neutrophil-neutrophil adhesion is mediated by L-selectin on one cell and PSGL-1 on the adjacent cell.  This results in strings of rolling neutrophils aligned along the direction of shear flow, which is thought to be relevant in the pathogenesis of VOC.  Cell aggregation resulting in microvascular congestion is also mediated by neutrophil-platelet interactions.  Activated platelets express P-selectin and can bind free-flowing neutrophils; thus, amplifying the surface area and the likelihood of capture on inflamed endothelium or on adherent neutrophils.  Adherent platelets aid in orchestrating diapedesis of neutrophils by Mac-1 binding to platelet GPIIb/IIIa in a fibrinogen-dependent manner.  Cellular cross-talk between platelet P-selectin binding to neutrophil PSGL-1 and Mac-1/fibrinogen/GPIIb/IIIa supports the efficient trafficking of neutrophils to sites of inflammation.  Taken together, this implicates Mac-1 and P-selectin as adhesion receptors that may play an important role in the heterotypic platelet adhesive events that occur down-stream of E-selectin in the progression of VOC.  Furthermore, these researchers stated that while the importance of selectins and their ligands could not be over-stated, there are key differences in function between human and mouse models that must be appreciated in order to efficiently develop effective therapeutics to prevent VOC.  The authors concluded that targeting these earliest selectin-dependent events in neutrophil recruitment with antagonists that block both initial tethering and signaling of adhesion appeared to be effective in mitigating VOC and painful episodes.  Early trials were promising and should be accompanied by further experimental studies including novel appropriate murine in-vivo models that address the various differences in the regulation of selectin function between mouse and human and ex-vivo analysis of treatment effectiveness in inflammatory recruitment especially of neutrophils and sickle red blood cells (sRBCs) over the course of the disease.  Taken together, this will shed new light on the role of selectins in the pathogenesis of VOC and hopefully contribute to establishing and refining a potentially beneficial selectin blocking strategy for the treatment of patients with SCD.

Hines et al (2021) noted that blood cell adhesion to P-selectin and vascular cell adhesion molecule-1 (VCAM-1) contributes to the pathophysiology of VOC events in patients with SCD.  In a single-center study, these investigators examined the use of standardized flow adhesion biomarkers in a 6-month, 35-subjects longitudinal study (The ELIPSIS Trial).  Flow adhesion of WB on P-selectin (FA-WB-Psel) and VCAM1 (FA-WB-VCAM), and of isolated white blood cells (WBCs) on P-selectin (FA-WBC-Psel) and VCAM-1 (FA-WBC-VCAM) were elevated on VOC days compared with non-VOC days, but only FA-WB-Psel reached statistical significance (p = 0·015).  Optimal cut-off values were established with Cox regression models for FA-WB-Psel (46 cells/mm²; hazard ratio (HR): 2.3; 95 % confidence interval [CI]: 1.4 to 4.0; p = 0.01) and FA-WB-VCAM (408 cells/mm², HR:1.8; 95 % CI: 0.9 to 3.45; p = 0.01).  A combined (FA-WB-Psel and FA-WB-VCAM) multi-marker risk score was also significantly (p = 0·0006) correlated with VOC risk that was 2-fold higher for intermediate and 5.64-fold higher for high score.  The concordance (C)-index for the multi-marker score was 0.63 in the 6-month period (95 % CI: 0.56 to 0.70), indicating a better ability to distinguish patient risk of VOC, compared to individual biomarkers FA-WB-VCAM (C-index: 0.57; 95 % CI: 0.49 to 0.65) or FA-WB-Psel (C-index: 0.58; 95 % CI: 0.53 to 0.62).  The authors concluded that the presented multi-marker score could be used to risk-stratify individuals with SCD during their steady state into low-, intermediate-, and high-risk strata for self-reported VOCs.  Such risk stratification could aid in focusing healthcare resources more efficiently to maintain health, personalize treatment selection to each patient's individual needs, and potentially reduce healthcare costs.  Moreover, these researchers stated that studies are under way to examine the ability of assays such as FA‐WB‐Psel to monitor response to SCD‐modifying therapies (e.g., crizanlizumab, L‐glutamine, voxelotor).

The authors stated that among the drawbacks of this trial, the most significant was that it was a single‐center study with a relatively low number of enrolled subjects (n = 35).  Furthermore, the described multi-marker score model was built based on the same data-set used for determination of the optimal cut‐off values; therefore, its reliability and performance should be re‐examined via independent external validation.  A greater degree of confidence in the discriminatory results between low-risk and high-risk VOC in SCD subjects could be achieved with a larger sample size, ideally with subjects recruited at multiple sites for higher diversity.  Another drawback was that the steady state samples were collected at a 3‐week interval; therefore, the scale and the rates of elevation of flow adhesion values before the VOC could not be precisely evaluated.

White et al (2022) stated that SCD is characterized by frequent and unpredictable VOC; sRBCs contribute to VOCs by participating in a series of adhesive events with blood cells and the vascular endothelium.  Adhesion assays have been used to examine the relationship between sRBCs adhesion and SCD severity.  These researchers developed a standardized, clinical flow adhesion assay of WB to vascular cell adhesion molecule (FA-WB-VCAM).  They examined the variability and clinical predictive value of FA-WB-VCAM in a 6-month longitudinal, observational study (The ELIPSIS Trial) in SCD patients during at-home, steady-state and self-reported VOCs, and following VOC resolution.  These investigators observed a strong relationship between FA-WB-VCAM and SCD severity.  Adhesion indices were significantly lower in SCD subjects on hydroxycarbamide and increased during VOCs; at-home VOCs had significantly higher FA-WB-VCAM than steady-state and contact VOCs.  SCD patients with a high frequency of self-reported VOCs had a pro-adhesive phenotype at steady state and were stratified into a high-adhesive phenotype cohort; 2 years prospectively these researchers observed a higher frequency of VOCs in the high-adhesion cohort.  The authors concluded that the findings of this study supported stratifying SCD patients based on steady-state FA-WB-VCAM and suggested that FA-WB-VCAM may be a plausible surrogate endpoint for SCD severity.  Moreover, these researchers stated that additional studies in a larger prospective cohort were underway to definitively establish the predictive value of FA‐WB‐VCAM for impending VOCs, identifying SCD patients with severe disease phenotypes, and to establish its role in therapeutic monitoring of SCD‐modifying therapies.  The authors stated that the principal drawback of this trial was that the study was not designed to collect information on acute interventions or to ensure blood was consistently taken before interventions; thus, a future study will determine this definitively.

Man et al (2023) noted that neutrophil recruitment to the inflamed endothelium is a multi-step process and is of utmost importance in the development of the hallmark VOC in SCD.  However, there is a lack of a standardized, clinically feasible approach for evaluating neutrophil recruitment to the inflamed endothelium for individualized risk stratification and therapeutic response prediction in SCD.  These researchers described a micro-fluidic device functionalized with E-selectin, a critical endothelial receptor for the neutrophil recruitment process, as a strategy to examine neutrophil binding under physiologic flow in normoxia and clinically relevant hypoxia in SCD.  They demonstrated that hypoxia significantly enhances neutrophil binding to E-selectin and promotes the formation of neutrophil-platelet aggregates.  Moreover, these investigators identified 2 distinct patient populations: a more severe clinical phenotype with elevated lactate dehydrogenase (LDH) levels and absolute reticulocyte counts, but lowered fetal Hb levels associated with constitutively less neutrophil binding to E-selectin.  Mechanistically, these researchers showed that the extent of neutrophil activation correlates with membrane L-selectin shedding, leading to the loss of ligand interaction sites with E-selectin.  They also revealed that inhibition of E-selectin significantly decreased leukocyte recruitment to activated endothelial cells.  The authors concluded that these findings suggested that a well-controlled, standardized flow adhesion assay enabled by the E-selectin micro-fluidic device would provide valuable information for the development of a personalized therapeutic strategy (e.g., an inhibitor targeting at E-selectin) that could aid in guiding therapeutic decisions for SCD patients.

The authors stated that this study had several drawbacks.  First, the study population with homozygous SCD (HbSS) was relatively modest.  A larger cohort will allow these investigators to better examine the associations between neutrophil recruitment and patient clinical phenotypes, as well as treatments such as transfusion and the use of hydroxyurea.  Second, increased L-selectin shedding on the surface of neutrophils is concordant with up-regulated αMβ2 integrin expression.  To link patient clinical phenotypes to neutrophil activation and reduced binding to E-selectin, flow cytometry characterization of L-selectin and αMβ2 integrin expression, as well as neutrophil adhesion to intercellular adhesion molecule 1 (ICAM-1) among test subjects will need to be examined.  Third, the mechanistic studies were carried out using blood samples from patients with SCD who have an inherently larger variation in neutrophil activation than healthy samples.  Fourth, the mechanism of neutrophil aggregation mediated by P-selectin or L-selectin-PSGL-1 interactions under hypoxia and its association with patient clinical phenotypes in SCD was not analyzed and warrants future investigations.

Gurkan et al (2024) stated that microfluidic lab-on-a-chip technologies enable the analysis and manipulation of small fluid volumes and particles at small scales and the control of fluid flow and transport processes at the micro-scale, resulting in the development of new methods to address a broad range of scientific and medical challenges.  Microfluidic and lab-on-a-chip technologies have made a significant impact in basic, pre-clinical, and clinical research, especially in hematology and vascular biology due to the inherent ability of microfluidics to mimic physiologic flow conditions in blood vessels and capillaries.  With the potential to significantly impact translational research and clinical diagnostics, technical issues and incentive mismatches have stymied microfluidics from fulfilling this promise.  These researchers described how accessibility, usability, and manufacturability of microfluidic technologies should be improved and how a shift in mindset and incentives within the field is also needed to address these issues.  They discussed the state of the microfluidic field regarding current limitations and propose future directions and new approaches for the field to advance microfluidic technologies closer to translation and clinical use.  The authors noted that while this report focused on using blood as the prototypical biofluid sample, the proposed ideas and research directions could be extrapolated to other areas of hematology, oncology, biology, and medicine.  Moreover, these researchers stated that the ability to predict sickle cell pain crises is an unmet need and opportunity for the microfluidics field.  Smart, low-cost microfluidics applications have been developed with successful proof-of-concept (POC) prototypes (e.g., smartphone microscopy, microflow cytometry, flow assay, and mobile ELISA).

Furthermore, UpToDate reviews on “Overview of the management and prognosis of sickle cell disease” (Field and Vichinsky, 2024), “Diagnosis of sickle cell disorders” (Vichinsky, 2024a), “Overview of the clinical manifestations of sickle cell disease” (Vichinsky, 2024b), and “Overview of compound sickle cell syndromes” (Vichinsky, 2024c) do not mention the use of microfluidic flow adhesion assay/test as a management tool.

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References