Phagocytosis: Laboratory Assessment of Immune Cell Function

Phagocytosis is not a single reaction, but a complex chain of events in innate immunity. It includes microbe recognition, attachment, cell exit from the vessel into the inflammatory site, engulfment, phagosome formation, fusion with granules, and subsequent intracellular destruction involving reactive oxygen species and enzymes. Therefore, phagocytosis testing cannot be reduced to a single, universal test: a defect can occur at any of these stages. [1]

In clinical immunology, "phagocytosis testing" most often refers to the evaluation of neutrophil function, and less commonly, monocyte function. In practice, physicians seek an answer not to the abstract question of "is the immune system functioning properly?" but to a much more specific one: whether there is an innate defect in the cells that are supposed to engulf and destroy bacteria and fungi, migrate to the site of inflammation, and generate an adequate innate response. [2]

Phagocytic defects account for approximately 10%-15% of primary immunodeficiencies. The most well-known include chronic granulomatous disease, leukocyte adhesion deficiency, Chédiak-Higashi syndrome, and cyclic neutropenia. However, it's not only rare congenital disorders that are clinically important: secondary neutrophil dysfunction also occurs in severe sepsis, malnutrition, certain drug exposures, and severe chronic inflammatory conditions. [3]

This is why modern diagnostics proceeds in stages. First, they evaluate the clinical picture, complete blood count, blood smear, and the presence of typical infections. Then they move on to more specific tests: respiratory burst assessment, adhesion molecule expression, sometimes direct absorption and bactericidal tests, and finally, molecular confirmation. [4]

The key practical conclusion that emerges from this is that phagocytosis testing is a panel approach, not a single "immune assay." The more accurately the physician understands which stage of the innate response is impaired—migration, oxidative burst, granular apparatus, or engulfment itself—the shorter the path to diagnosis and the lower the risk of misinterpretation. [5]

Phagocytic response stage	What can be violated	Which test is most important?
Adhesion to the endothelium and exit from the vessel	Deficiency of integrins and selectin ligands	Flow cytometry CD11a, CD11b, CD18, CD15s
Oxidative explosion	Defect of nicotinamide adenine dinucleotide phosphate oxidase complex	Dihydrorhodamine 123 test
Granular apparatus and intracellular processing	Primary granular defects	Smear, specialized immunological and genetic tests
Absorption and bactericidal activity	Functional neutrophil dysfunction	Whole blood phagocytosis and killing tests in specialized centers

Sources for the table: MSD Manual, ARUP, GeneReviews, current reviews on chronic granulomatous disease. [6]

When is phagocytosis testing really necessary?

The most typical indication is recurrent severe bacterial and fungal infections, especially deep abscesses, lymphadenitis, osteomyelitis, liver, lung, and skin infections. Phagocytic defects are particularly characterized by recurrent staphylococcal, gram-negative, and aspergillosis infections, as well as inflammatory granulomas and poor tissue healing.[7]

Age and type of manifestation are very important. If a child regularly experiences severe purulent infections in infancy or early childhood, and the pathogens are recurrent or unusual, the likelihood of a congenital phagocyte defect increases. Particularly concerning are infections that require intravenous antibiotics, are poorly responsive to standard treatment, and recur in multiple organs. [8]

A separate classic situation is suspected leukocyte adhesion deficiency. It is characterized by a delay in cord separation of more than 3 weeks, severe necrotic soft tissue infections, the absence of pus during infections, marked leukocytosis, and severe periodontitis. In such cases, the study of "phagocytosis" in the narrow sense should not be limited to functional tests: flow cytometry of adhesion receptors is given priority. [9]

Secondary conditions must also be considered. In severe sepsis, malnutrition, some inflammatory diseases, and in children with severe growth retardation, neutrophil function may be reduced or distorted without a congenital defect. This does not render the test useless, but it does change the interpretation: an abnormal result in this case does not always equate to primary immunodeficiency. [10]

Finally, phagocytic testing is indicated not only for the patient but also for family members. For chronic granulomatous disease and leukocyte adhesion deficiency, genetic counseling of relatives makes clinical sense, and in X-linked chronic granulomatous disease, a dihydrorhodamine 123 test can reveal a mosaic pattern in female carriers. [11]

Clinical situation	How relevant is the study?
Recurrent deep bacterial or fungal infections	Very appropriate
Abscesses of the liver, lungs, skin, lymph nodes	Very appropriate
Delayed cord separation and infection without pus	Particularly important for excluding leukocyte adhesion deficiency
Unexplained severe leukocytosis in an infant	Requires assessment of adhesion and migration defects
Severe sepsis and secondary immune dysfunction	Functional assessment is possible, but interpretation is more difficult
Family history of chronic granulomatous disease	Functional and genetic methods are needed

Sources for table: MSD Manual, GeneReviews, Immune Deficiency Foundation, 2024 Chronic Granulomatous Disease Review. [12]

What methods are used today?

The main modern method for assessing respiratory burst is the dihydrorhodamine 123 test. It is based on flow cytometry and measures the ability of neutrophils to oxidize dihydrorhodamine 123 to a fluorescent product after stimulation, usually with phorbol 12-myristate 13-acetate. Essentially, it is a functional surrogate for the activity of the nicotinamide adenine dinucleotide phosphate oxidase complex and the main screening test for chronic granulomatous disease. [13]

The nitroblue tetrazolium assay was historically the standard, but has now been replaced by the dihydrorhodamine 123 assay in terms of sensitivity, quantification, and reproducibility. However, it has not disappeared entirely: in resource-limited settings, it can still be used as a working diagnostic tool, although in laboratories with flow cytometry, it is considered a backup and less convenient option. [14]

If leukocyte adhesion deficiency is suspected, neutrophil surface phenotyping using flow cytometry becomes the key method instead of the respiratory burst test. For type 1, CD18 and associated CD11a and CD11b are primarily assessed, while for type 2, CD15s is additionally assessed. A decrease or absence of these molecules correlates well with impaired neutrophil migration and adhesion. [15]

Direct whole-blood phagocytosis and intracellular killing assays also exist. They typically use labeled bacteria or particles and allow for quantitative assessment of how many targets have been engulfed by neutrophils and how effectively they subsequently kill the microorganism. However, such methods have not become as universal as the dihydrorhodamine 123 assay and are more often used in research or highly specialized centers. [16]

Following functional testing, molecular confirmation is increasingly required. In chronic granulomatous disease with recurrent respiratory burst dysfunction, it is recommended to analyze at least CYBA, CYBB, NCF1, NCF2, NCF4, CYBC1, and RAC2. In leukocyte adhesion deficiency, genetics is particularly important for family counseling and definitive confirmation of the defect subtype. [17]

Method	What does it evaluate?	Where it is especially useful
Dihydrorhodamine 123 test	Neutrophil respiratory burst	Screening for chronic granulomatous disease, carriage, and post-transplant monitoring
Nitroblue tetrazolium test	Neutrophil oxidative response	A backup and historical method, sometimes useful when resources are limited
Flow cytometry CD18, CD11a, CD11b, CD15s	Neutrophil adhesion and migration	Diagnosis of leukocyte adhesion deficiency
Direct phagocytosis tests	Absorption of particles or bacteria	Auxiliary and specialized situations
Intracellular killing tests	The ability to destroy ingested microbes	Specialized centers and complex cases
Genetic testing	Causal molecular defect	Confirmation of diagnosis and family counseling

Sources for table: GeneReviews, ARUP, MSD Manual, Immune Deficiency Foundation, 2024 review. [18]

Why tests can be false or difficult to interpret

Pre-analysis is critical for the dihydrorhodamine 123 assay. ARUP specifies that live neutrophils, heparinized whole blood, and the sample must not be refrigerated or frozen, and many protocols require a concurrent control blood sample. If transportation and delivery times are compromised, the results become difficult to interpret or even unreliable. [19]

Even with a high-quality sample, biological pitfalls exist. ARUP reports describe an intermediate pattern between normal and abnormal fluorescence, which can occur during acute inflammation. In such situations, the laboratory itself recommends repeating the test after the inflammatory process has resolved, rather than drawing a final conclusion based on a single analysis. [20]

A separate problem is myeloperoxidase deficiency. It can produce a picture similar to chronic granulomatous disease, especially if one relies solely on the dihydrorhodamine 123 test. Modern reviews emphasize that these conditions must be distinguished: in chronic granulomatous disease, the respiratory burst is impaired in all phagocytes, whereas in myeloperoxidase deficiency the picture is already biologically different. [21]

There are also drug-related interferences. A 2024 review notes that false-positive results on respiratory burst function tests are possible in patients receiving paracetamol, metamizole, or mesalazine. This is a rare but crucial detail: if primary immunodeficiency is suspected, without taking into account the drug history, a false diagnostic path can be taken. [22]

Finally, the clinical context itself influences interpretation. Severe neutropenia can render functional tests unreliable, sepsis can paradoxically alter various neutrophil functions in different directions, and secondary immunodeficiencies often produce a mixed picture. Therefore, a phagocytosis study is always a contextual analysis, not a laboratory "diagnostic seal."

Reason for distortion	How does it manifest itself?	What to do
Incorrect transportation or cooling of the sample	Falsely decreased neutrophil function	Repeat the study with proper logistics
Acute inflammation	Intermediate pattern of the dihydrorhodamine 123 test	Repeat the analysis after the inflammation has resolved.
Myeloperoxidase deficiency	Simulation of respiratory burst defect	Additional examination for myeloperoxidase
Paracetamol, metamizole, mesalazine	Possible false-positive functional results	Take into account the drug history
Severe neutropenia	Technically unreliable test	First, estimate the absolute neutrophil count
No control sample	Difficult interpretation of abnormal result	Compare with control blood from the same day

Sources for table: ARUP, 2024 review of chronic granulomatous disease diagnostics. [24]

What diseases are most often searched for using these tests?

The most common target diagnosis is chronic granulomatous disease. This is a congenital defect of the nicotinamide adenine dinucleotide phosphate oxidase complex, in which phagocytes are unable to generate reactive oxygen species. Clinically, the disease causes recurrent severe bacterial and fungal infections, especially of the skin, lungs, liver, lymph nodes, and bones, as well as granulomatous inflammation. [25]

The second most important defect is leukocyte adhesion deficiency. Here, the problem is not the respiratory burst, but rather the inability of neutrophils to properly adhere to the endothelium, exit the vessel, and enter tissue. Clinical clues are classic: delayed cord separation, marked leukocytosis, soft tissue infections without pus formation, poor wound healing, and severe periodontitis. [26]

Chédiak-Higashi syndrome is a rarer but fundamentally important variant. It is characterized by abnormalities of the phagocyte granular apparatus, recurrent infections, oculocutaneous albinism, hepatosplenomegaly, lymphadenopathy, neurological manifestations, and a tendency to bleed. A standard "phagocytosis test" is rarely sufficient in this case, and a blood smear, granule morphology, and genetics play a significant role. [27]

It's important to remember that not every abnormal result indicates a congenital immunodeficiency. Secondary neutrophil dysfunction can occur with severe infections, malnutrition, some drug exposures, and chronic inflammation. Therefore, an abnormal DHR, reduced bactericidal activity, or atypical neutrophil behavior without the characteristic clinical features of the primary defect should prompt consideration of secondary causes. [28]

In practice, phagocytosis testing is particularly valuable because it helps narrow the differential diagnosis. It distinguishes oxidative burst defects from migration defects, and true congenital syndromes from secondary immune dysfunction. However, a definitive diagnosis almost always requires a combination of clinical examination, functional testing, phenotyping, and genetic confirmation. [29]

Disease	What is broken?	The most characteristic laboratory accent
Chronic granulomatous disease	Respiratory burst	Pathological test with dihydrorhodamine 123
Leukocyte adhesion deficiency type 1	CD18 and associated integrins	Decreased CD11 and CD18 on neutrophils
Leukocyte adhesion deficiency type 2	Fucosylation and CD15s	Reduced or absent CD15s
Chediak-Higashi syndrome	Granules and lysosomal traffic	Morphological and genetic characteristics, not one universal functional test
Myeloperoxidase deficiency	Pseudo-defect in some functional tests	Differentiation with chronic granulomatous disease is necessary.
Secondary neutrophil dysfunction	The function is impaired for the second time	The result is read only in a clinical context.

Sources for the table: MSD Manual, GeneReviews, ARUP, 2024 review. [30]

What to do after an abnormal result

If an abnormal result is obtained for the first time, the first step is to check the reliability of the test itself. It's important to determine whether the sample was delivered on time, whether there was acute inflammation, whether there were sufficient neutrophils, whether a control sample was available, and whether the patient was taking medications that could interfere with the result. Without this, it's easy to start expensive and worrying follow-up investigations on the wrong track. [31]

If the abnormal result concerns the respiratory burst and is confirmed repeatedly, the next step is molecular verification of chronic granulomatous disease and related defects. A current review recommends testing at least seven genes associated with the nicotinamide adenine dinucleotide phosphate oxidase complex for recurrent abnormalities. This is no longer just a diagnostic step, but a decision on long-term management for the patient and family. [32]

If the problem lies in the adhesion and migration zone, an abnormal CD11, CD18, or CD15s panel result requires confirmation and rapid reassignment. In severe leukocyte adhesion deficiency, the mainstay of treatment remains prophylactic antibiotics, sometimes granulocyte transfusions, and hematopoietic stem cell transplantation is considered the only truly effective and potentially curative method. For type 2, fucose correction may be considered. [33]

For chronic granulomatous disease, once the diagnosis is confirmed, treatment goes far beyond the laboratory. Current reviews recommend antibacterial and antifungal prophylaxis, reduced exposure to mold, and regular specialized monitoring. Hematopoietic stem cell transplantation is now considered a potentially curative option, not just a "last resort" for extremely severe cases. [34]

If the result is borderline or inconsistent with the clinical picture, it's wiser not to rush into rare diagnoses and instead reassess the entire immunological context. Sometimes, after a normal dihydrorhodamine 123 test and adhesion panel, it's necessary to return to more basic tests: absolute neutrophil count, blood smear, immunoglobulins, antibody responses to vaccines, the complement system, and secondary causes of immunodeficiency. This approach is often the most effective way to avoid missing the true cause of infections. [35]

Situation after the result	The next smart step
Borderline test with dihydrorhodamine 123 in the presence of acute inflammation	Repeat the study outside the acute phase
Repeat pathological test with dihydrorhodamine 123	Genetic confirmation of nicotinamide adenine dinucleotide phosphate oxidase complex defect
Decreased CD18, CD11, and/or CD15s	Confirm leukocyte adhesion deficiency and discuss specialized treatment
Normal dihydrorhodamine 123 test in severe infections	Look for other neutrophil, complement, antibody defects and secondary causes
Confirmed chronic granulomatous disease	Infection prevention, genetic counseling, transplant discussions
Confirmed leukocyte adhesion deficiency	Prevention, severity assessment, transplant routing

Sources for the table: MSD Manual, 2024 revision, ARUP, Immune Deficiency Foundation. [36]

FAQ

1. Are the phagocytosis assay and the dihydrorhodamine 123 test the same thing?
No. The dihydrorhodamine 123 test evaluates only one part of the phagocytic response—the neutrophil respiratory burst. The phagocytosis assay, as a broader concept, also includes adhesion, migration, engulfment, intracellular killing, and sometimes genetic confirmation of the defect. [37]

2. What test is currently considered the primary screening method for chronic granulomatous disease?
The dihydrorhodamine 123 flow cytometry test is considered the primary screening method. It is more sensitive and quantitatively informative than the historical nitroblue tetrazolium test. [38]

3. Is the nitroblue tetrazolium test completely obsolete?
Not entirely. In laboratories with available flow cytometry, it is inferior to the modern dihydrorhodamine 123 test, but in resource-limited settings, it can still be used as a working diagnostic tool. [39]

4. Why is a standard "phagocytosis test" insufficient when leukocyte adhesion deficiency is suspected?
Because the problem with this disease is primarily the disruption of adhesion and neutrophil release from the vessel, not the oxidative burst itself. Therefore, the CD11, CD18, and, if necessary, CD15s panels become key. [40]

5. Can acute inflammation interfere with the results?
Yes. Intermediate and difficult-to-interpret patterns have been reported for the dihydrorhodamine 123 test in the presence of acute inflammation, so laboratories often recommend repeating the test after the inflammation has resolved. [41]

6. What drugs can interfere with respiratory burst function tests?
A recent review identifies paracetamol, metamizole, and mesalazine as drugs that can produce false-positive results in respiratory burst function tests. [42]

7. What is myeloperoxidase deficiency and why is it important to remember?
This condition can mimic chronic granulomatous disease in terms of functional tests. Therefore, if the dihydrorhodamine 123 test result is abnormal, it is always important to remember to differentiate between these two conditions. [43]

8. Can a diagnosis be made based on just one abnormal result?
Usually not. The modern approach requires at least a repeat test if the results are questionable, clinical considerations, and sometimes neutrophil surface phenotyping and genetic confirmation. [44]

9. What if a child is frequently ill, but the dihydrorhodamine 123 test is normal?
A broader search is needed. If the respiratory burst is normal, it is worth assessing adhesion molecules, neutrophil count, blood smear, immunoglobulins, antibody responses to vaccines, the complement system, and secondary causes of immunodeficiency. [45]

10. What diseases are most often confirmed after such an examination?
Chronic granulomatous disease and leukocyte adhesion deficiency are most often looked for. Less frequently, Chédiak-Higashi syndrome, myeloperoxidase deficiency, and other rare neutrophil defects are detected. [46]