SEARCH

SEARCH BY CITATION

Keywords:

  • PNH;
  • flowcytometry;
  • CD55;
  • CD59 and FLAER

Abstract

  1. Top of page
  2. Abstract
  3. FLAER
  4. Haemolytic hiatus and transfusion requirement
  5. Diagnosis of PNH
  6. Bone marrow analysis
  7. Classification of PNH
  8. External quality assurance program diagnosis of PNH
  9. Conclusion
  10. References

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired clonal disorder of haematopoietic stem cells. The molecular defect in PNH is mutation in the phosphotidylinositol glycan complementation class A (PIGA gene) causing defect in glycosylphosphatidylinositol anchored proteins (Cell, 73, 1993, 703). The deficiency of these GPI-anchored proteins on the membranes of haematopoietic cells lead to the various clinical manifestations of PNH. Clinically PNH is classified into classic PNH, PNH in the setting of another specified bone marrow disorder and sub clinical PNH. Size of the PNH clone differs in these different subtypes. The management of PNH has been revolutionized by the advent of monoclonal antibody, eculizumab. Thus, today it is important to have sensitive tests to diagnose and monitor the clone size in patients of PNH. Before 1990, diagnosis of PNH was made using complement based tests. However in the last decade, flowcytometry has become the gold standard diagnostic test as it has increased sensitivity to detect small clones, ability to measure clone size and is not affected by blood transfusions. This review is aimed to focus mainly on the different methods available for the detection of PNH clone and the recent advances and recommendations for the flowcytometric diagnosis of PNH.

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare form of acquired haemolytic disorder characterized, in classic cases, by attacks of intravascular haemolysis and haemoglobinuria (1). Other common clinical manifestations include thrombocytopenia, leucopenia or both and recurrent venous thrombosis involving unusual sites (2). It results from the expansion of an abnormal clone of haematopoietic stem cells harboring somatic mutation in phosphotidylinositol glycan complementation class A (PIGA) gene, which has a survival advantage in the setting of a specific type of bone marrow injury (putatively immune) (3). The PIGA gene maps to Xp22.1; therefore only one allele of PIGA is transcribed in both sexes because of X inactivation. The mutation results in a deficiency in the surface expression of all GPI-anchored proteins (GPI-AP) because of defective synthesis of glycosylphosphatidylinositol (GPI) in the endoplasmic reticulum. Over 20 other genes involved in GPI production have now been described. However in all reported cases of acquired PNH, the mutation is detected only in the PIGA gene. The PNH stem cell and all of its progeny have either a complete (Type III cells) or partial deficiency (Type II cells) of an entire class of GPI-AP depending on the type of mutation in PIGA gene (4, 5). These proteins serve many purposes and function as enzymes, receptors, complement regulators, and adhesion molecules (6). Among these proteins, CD55 (decay accelerating factor) and CD59 (membrane inhibitor of reactive lysis) have been identified as playing an important role in the regulation of complement activation by preventing it from being attached to cell membranes. The deficiency of these proteins leads to an increased susceptibility of the PNH cells to complement-mediated cell lysis (7).

Two rare inherited genetic conditions have been described that share some of the clinical features characteristic of patients with PNH. One is a complete deficiency of CD59 resulting from the mutation in the CD59 gene, presenting with attacks of complement-mediated haemolysis and arterial thrombosis (8). The expression of the other GPI-AP was normal in this patient. The second form was described in two families with inherited deficiency of GPI anchor synthesis resulting from a mutation in the promoter region of the PIGM gene. It was inherited in an autosomal recessive manner and was characterized by a propensity to venous thrombosis and seizures without overt intravascular haemolysis (9). The affected patients showed a very peculiar phenotype with all leukocytes showing GPI-deficient cell populations as seen in classical PNH (10). However, the classical red cell pattern of clearly definable type II and/or type III red cell populations was not seen, and a spectrum of weak GPI antigen expression was noted. Bone marrow failure, which is a central component of PNH, was absent in individuals with both these conditions.

Diagnosis and screening of PNH has improved considerably with the development of flow cytometry-based testing using many of the widely available clustered monoclonal antibodies to GPI-AP. Multicolor flow cytometry in particular provides a powerful tool for rapid, sensitive, and specific screening and diagnosis of PNH. This review will focus on the different tests used for detection of PNH clone, and recent recommendations for its diagnosis.

Detection of PNH clones

There are various modalities which are currently being used for the diagnosis of PNH. These include complement based tests and sephacryl gel card techniques (GCT), both of which can detect PNH positive red blood cells (RBC), flowcytometry based assays which can detect PNH clone in all the peripheral blood (PB) cells and molecular diagnostic tests for detection of mutations in PIGA gene (Table 1).

Table 1.   Laboratory tests for the diagnosis of PNH
Laboratory testsPrincipleAdvantagesDisadvantages
  1. PNH, paroxysmal nocturnal haemoglobinuria; RBC, red blood cells; GPI-AP, GPI-anchored proteins; AA, aplastic anemia; FLAER.

Ham test Sucrose lysis testThe lysis of PNH red blood cells exposed to activated complementCheap and simple to performLabor intensive, low sensitivity and specificity, not quantitative
Sephacryl gel card testHaemagglutinin test using the gel microtyping systemCheap and simple to performLow sensitivity and not quantitative
Flow cytometric analysis  CD 59 and CD55 on RBCStudy expression of GPI-AP using monoclonal antibodies by flowcytometryRapid, sensitive, and quantitativeLower estimate of clone size because of shorter life span of RBC
 CD 59, CD55, CD24, CD66b, and CD16 for granulocytesBetter estimate of clone sizeRequire fresh blood sample Difficult in patients with AA with neutropenia
 FLAER for granulocytesLack of FLAER binding to PNH granulocytesHighly sensitive as single reagent for diagnosis

Complement based tests

Traditionally tests used for diagnosis of PNH were dependent on the complement sensitivity of the RBC. The commonly used complement based assays are HAM’s and Sucrose lysis test. These tests, although suitable for haemolytic PNH, cannot reliably detect small populations of affected red cells. The detection limit of these tests is between 4.2% and 5% (10). Moreover, they are not specific for the diagnosis of PNH, as the HAM’s test can also be positive in HEMPAS variety of congenital dyseryhtropoietic anemia and the Sucrose lysis test can be positive in other conditions like megaloblastic anemia, autoimmune haemolytic anemia, etc. The complement lysis sensitivity test, which was developed by Rosse and colleagues in the 1960s (11, 12), can provide a more accurate assessment of the proportion and type of PNH red cells. Unfortunately, this test is extremely laborious and difficult to perform and is not applicable to routine diagnostic use.

GCT

This test is based on the principle of haemagglutination following an antigen–antibody reaction. The commercially available GCT detects RBC population deficient in CD55 and CD59. Its sensitivity has been reported to vary from 2 to 10% of type III erythrocytes (13) and this is similar to complement based assays (14). As it is easy to perform and interpret it has replaced complement based assays in many laboratories which do not have the facility for flowcytometry. However, there are certain limitations of this method. It is not quantitative, not sensitive for detection of small clones, and can not detect the GPI deficient granulocytes in PNH patients.

Flowcytometry testing

Flowcytometry acts as a rapid, sensitive and reproducible diagnostic tool for the detection of PNH clones in different PB cell populations. It was first described in 1985 (15, 16) and has now become the ‘Gold Standard’ for diagnosis of PNH. Flowcytometry can identify, quantitate, and subtype (Type I, Type II, and Type III cells) populations of these GPI-AP deficient cells.

Detection of PNH clones by flowcytometry can be done by studying the expression of GPI-AP on RBC and PB leukocytes using monoclonal antibodies specific for these proteins or by studying the GPI anchor itself, using FLAER.

A large number of GPI-AP have been described till now. Their expression varies greatly within the different subpopulations of haematopoietic cells (17) and also with stage of maturation of the cells (18). Hence for the selection of antibodies and correct interpretation of results, it is essential to have a detailed knowledge of the cellular distribution of GPI-AP and their expression at the different stages of haematopoietic cell differentiation (19).

Apart from antibody selection, there are a number of important considerations in terms of methods of sample processing, cell populations to be studied and gating strategies for the accurate analysis of GPI-AP.

Analysis of RBC

Expression of GPI-AP has been extensively studied on RBC mainly because haemolysis is an important clinical manifestation of the disease and the initial experiments were focused on demonstration of deficient RBC. The recommended approach for routine screening of PNH is a simultaneous evaluation of two GPI-linked antigens (CD55 and CD59) using directly conjugated monoclonal antibodies. For establishing the diagnosis, the cells must be deficient for both the GPI-AP. This is because rare hereditary deficiencies of the individual complement regulatory molecules CD55 and CD59 can occur. In contrast to the majority of PNH cases, all cells in these patients are either CD55 or CD59 deficient.

Adequate dilution of sample and titration of antibodies is essential while standardizing the staining of RBC for CD55 and CD59. There may be a significant technical problem of agglutination while staining RBC with two or more monoclonal antibodies. This can be minimized by adequately diluting the cells and adding a wash step with phosphate buffered saline (PBS) before staining.

There is no consensus on the gating strategies which can be used for RBC. Glycophorin A (CD234a) is one of the most commonly used antibodies for this purpose. However in a multicolor panel it can cause problem of red cell agglutination. Hence a commonly applied strategy is the gating based on the physical characteristics of RBC using forward scatter (FSC) and sideways scatter (SSC) amplification in log mode (19).

Analysis of red cells in an untransfused PNH patient provides the clearest definition of type III (complete deficiency), type II (partial deficiency), and type I (normal expression) populations. However, a marked variation in the distributions of these sub-populations is seen from patient to patient and the separation between the types of cells is not always clear cut. In addition, the intensity of expression of CD55 and CD59 on RBC is not same. CD59 expression is stronger than CD55 and hence CD59 gives much better separation of Type I, II, and III cells than CD55 (Fig. 1). A distinct separation of Type I, II, & III can be achieved by washing cells twice after staining which removes excess of antibodies from the sample. Another major advantage of analysis on the RBCs is that it can be performed on a sample stored up to 21–30 d at 4–8°C (19).

image

Figure 1.  Expression of the GPI-linked antigens on RBC in patient with paroxysmal nocturnal haemoglobinuria (PNH). (A) Gating of RBC using forward scatter (FSC) and sideways scatter (SSC) amplification in log mode. (B) Histogram showing expression of CD59 on RBC with clear separation of Type I, II and III cells. (C) Histogram showing expression of CD55 on same patient with poor separation between Type I, II, & III cells compared to CD59.

Download figure to PowerPoint

The total clone size on RBC is always less than that seen on neutrophils. This may be because of lysis of Type III RBC and presence of normal transfused RBC, circulating in the patient. Even the proportion of Type I, II, and III cells may differ between the neutrophils and the RBC in the same patient. This is best documented in Fig. 2. Recent red cell transfusion is unlikely to obscure the diagnosis of PNH, but the flow cytometric histogram will be affected, as transfusion will increase the proportion of cells with normal expression of CD55 and CD59. Therefore, to obtain accurate information about the percentage of GPI-AP-deficient erythrocytes, analysis ideally should be performed prior to transfusion or during a period of transfusion abstinence (at least 1 month, but longer if clinically safe). It is shown that clone size estimation on reticulocytes may give a better estimate of the former. This can be studied using Thiazole orange and CD59 and is shown to correlate well with granulocyte clone size (20). However, the clinical utility of this test is not yet established.

image

Figure 2.  PNH clone size and the proportion of Type I, II, and III cells differs on neutrophils and RBC done simultaneously on the same patient. (A) Shows analysis of granulocyte with FLAER showing predominantly Type III cells. (B) Shows analysis on RBC with CD59 on the same sample showing markedly reduced Type III cells and higher proportion of Type II cells.

Download figure to PowerPoint

Analysis of granulocytes

Analysis of expression of GPI-AP on granulocytes provides additional clinically relevant information. In contrast to GPI-AP-deficient red cells, the life span of PNH granulocytes is normal. Therefore, the proportion of abnormal granulocytes more accurately reflects the PNH clone size and is unaffected by red cell transfusion. However, standardization of granulocytes staining is technically more difficult. The sample preparation protocol has a significant impact on the quality of the staining for the CD55 and CD59 antigens on granulocytes. There are various methods which have been described for this purpose (i) Lyse-wash-stain technique, (ii) Stain-lyse-wash technique, and (iii) Stain-no lyse-no-wash technique (21).

Lyse-wash-stain technique: This is the most commonly used method for granulocyte staining. Briefly, 50–100 μL of anticoagulated peripheral blood sample is taken. RBCs are lysed using ammonium chloride based lysing reagent. The leukocytes are then washed with PBS and incubated with titrated amount of antibodies for 15–30 min at room temperature in the dark. The excess of antibody is then washed with PBS and cells are re-suspended in 500 μL of PBS for analysis. This method gives the best pattern of antigen expression (21) and requires less amount of antibodies compared to the other two methods. However, it is important to note that the lysing solution used should not contain fixative. Fixative if present may lead to non-specific binding of antibodies to the neutrophils and therefore the PNH clone may be totally missed.

Stain -lyse-wash technique: In this method cells are stained by adding titrated amounts of antibodies to 25–100 μL of whole blood, and incubating for 15–30 min at room temperature in the dark. RBCs are then lysed using lysing reagent and leukocytes are washed with PBS and re-suspended with 500 μL of PBS for analysis. The large amount of RBCs and platelets present can interfere with the staining and one requires large amount of antibodies for optimum results.

Stain-no lyse-no-wash technique: In this method 3 μL of blood is incubated with 10 μL of monoclonal antibodies for 30 min. It is then re-suspended in 500 μL of PBS for analysis. This method has also shown optimal patterns of antigen staining for CD55 and CD59 (21). In addition, the procedure allows simultaneous analysis of CD55 and CD59 expression on red cells, platelets, neutrophils, monocytes, and lymphocytes if combined with CD45, CD61, and CD64. However, in our experience PNH clone has often been missed using this technique. This may be because of the excess of antibody non-specifically binding the neutrophils. In addition, one may not be able to acquire adequate neutrophils especially in patients with neutropenia as only 3 μL sample is used for the procedure.

Another important issue during analysis of granulocytes is the gating strategy that should be used. Most of the previous studies were based on the FSC/SSC gating on granulocytes, but this is often not sufficient when analyzing samples with low neutrophil counts and very small clone sizes. In addition, in myelodysplastic syndrome (MDS) the neutrophils are often hypogranular and one may require a non-GPI linked lineage marker/SSC approach. CD15, CD33, and CD45 are some of the useful antibodies for this purpose.

Unlike RBC, analysis on granulocytes needs to be carried out within the first few hours of collection. As the sample ages, the autofluorescence and the non-specific binding of the granulocytes to the antibodies increases. Use of appropriate negative isotype controls under such situations may be helpful (19). It is often difficult to achieve clear separation of Type I, II, and III cells on granulocytes. In addition, in PNH patients, type I cells may sometimes be absent making it difficult to define the limits for Type I and Type II cells. It is useful to run a normal control in such situations. This not only helps to set the cut off limit but also acts as a quality check.

Intensity of expression of CD55 is better than CD59 on granulocyte as against RBC (17). Hence reliable separation of Type I, II, and III population is better achieved by CD 55 (Fig. 3). Antibodies such as CD16, CD24, and CD66b work equally well for granulocyte analysis. Combination of CD16/CD66b has the added advantage of clearly separating granulocytes from eosinophils and is shown to be superior to CD55/CD59 in screening for sub clinical PNH (22). CD16 and CD66b are expressed late in myeloid maturation, and their detection enables a clear resolution between PNH cells (negative populations) and normal granulocytes (positive populations). However, one has to keep in mind that CD16 if used in isolation can cause confusion and potential misdiagnosis. An infrequent polymorphism in the CD16 determinant renders the antigen undetectable to some monoclonal antibodies and not others (19). In addition, samples with proportionately high eosinophils will have a large CD16-negative component, as this antigen is not constitutively expressed on eosinophils.

image

Figure 3.  Expression of CD55, CD59 and FLAER on neutrophils in a patient with PNH. (A) Shows gating of neutrophils using SSC/CD45 gating strategy. (B) Histogram showing expression of CD55 on neutrophils with Type I, II, and III cells. (C) Histogram shows expression of CD59 on same patient showing poor separation between Type I, II, & III cells compared to CD55. (D) Histogram shows FLAER expression with clear separation of Type I, II, & III cells.

Download figure to PowerPoint

Analysis of monocytes

Analysis on monocytes is technically difficult as absolute number of monocytes is generally low in PNH patients thus making them difficult to gate. CD14, CD55 are found to be better compared to CD59 for their analysis. Use of CD33, CD4, or CD64 in a multicolor analysis helps to identify the monocyte population more accurately as compared to FSC/SSC gating. Monocyte and granulocyte clone sizes correlate well with each other as they both are derived from a common progenitor.

Analysis of lymphocytes

Lymphocytes have a prolonged life span and only those arising postdisease onset will be deficient in GPI-AP. Therefore majority of them will express these proteins even in the presence of PNH and hence lymphocyte analysis alone is not recommended for diagnosis.

Analysis of platelets

There appears to be no clear consensus in the literature on the immunophenotypic analysis of platelets in PNH. The fluorescent intensity of CD59 and CD55 expression on normal platelets appears to be weak, with up to 10% of them not expressing these two antigens (19). Whether this is genuine or a reflection of an insensitive technique remains unclear. As a consequence, populations of GPI-deficient platelets in PNH patients are not easily distinguished. The diagnostic utility and clinical relevance of examining platelets in PNH patients has not been established.

FLAER

  1. Top of page
  2. Abstract
  3. FLAER
  4. Haemolytic hiatus and transfusion requirement
  5. Diagnosis of PNH
  6. Bone marrow analysis
  7. Classification of PNH
  8. External quality assurance program diagnosis of PNH
  9. Conclusion
  10. References

An alternative flowcytometric approach that makes it possible to perform less extensive testing for the diagnosis of PNH has recently been suggested (23). This assay utilizes Aerolysin, the toxin of the bacterium Aeromonas hydrophila, which binds directly to the GPI anchor. It is secreted as an inactive protoxin, proaerolysin, that is converted to the active form, through proteolytic removal of a C-terminal peptide (24). Aerolysin, thus generated binds to cell surface structures and oligomerizes, forming channels that result in cell lysis. Initially, this reagent was used to demonstrate the resistance of PNH erythrocytes to aerolysin and also to enrich GPI-negative PNH cells (25). Two point mutations were introduced to obtain a protein that still binds GPI upon activation but lacks lytic activity. By coupling this mutant proaerolysin to a fluorescent marker (Alexa Fluor 488), a reagent (FLAER) was produced that stains cells containing GPI proteins but not PNH cells lacking GPI (25). As this reagent detects the GPI anchor itself, it can be used to investigate all peripheral blood cell types except erythrocytes, which do not express the necessary activating proteases.

FLAER as a single agent in a simple and fast whole blood staining procedure is shown to be equally effective as compared to conventional immunophenotyping (26). Although its use is restricted to leukocytes, it is proving to be particularly useful for reliable quantification of small PNH clones. FLAER is more sensitive than single parameter (CD55 or CD59) analysis on granulocytes because of its high signal to noise ratio (27) and gives better separation of Type I, II, and III cells (Fig. 3). Combining FLAER with multiparametric flowcytmometry can further improve the sensitivity and specificity of the test (27). Although PNH is a rare disease, it is one of the most frequently requested tests in a haematology laboratory. Hence FLAER as a single agent or in combination with one non-GPI-linked antibody can be used as a very sensitive screening test; positive cases can then be confirmed by multiparametric flowcytometry, thus saving on the expensive resources. In addition, it is a robust assay and can be performed on samples stored up to 24–48 h; this is a distinct advantage over the CD55 and CD59 based assays, which need to be performed within 8 h of collection (27). The advantages and disadvantages of this technique compared to standard immunophenotypic diagnosis of PNH are summarized in Table 2.

Table 2.   Comparison between FLAER and immunophenotyping for the diagnosis of PNH
  1. PNH, paroxysmal nocturnal haemoglobinuria; GPI-AP, GPI-anchored proteins; AA, aplastic anemia; MDS, myelodysplastic syndrome.

FLAERImmunophenotyping using monoclonal antibodies against GPI-AP
Sensitive as a single agent and hence more economical as screening testAt least two antibodies required
Detection of PNH clone only on leukocytesDetection of PNH clone on all peripheral blood cells
Better separation Type I, II, and III cells on granulocytesSeparation of Type I, II, and III cells on granulocytes is not always clear
Better estimation of clone size on granulocytes and monocytes and hence useful for estimation of small clone on granulocyte in AA and MDS using multiparametric assayEssential for estimation of clone size on RBCs and monitoring of RBC clone size in patients on Eculizumab therapy
More robust assay for detection of clone on granulocytes, can be performed on samples stored up to 48 hAnalysis on granulocyte needs to be performed within 8 h of collection, but analysis on RBCs can be done in samples stored up to 21–30 d

Analysis of RBC with CD59 is useful in monitoring patients on eculizumab therapy. Patients responding to therapy will show a progressive increase in the RBC clone because of prolonged survival of the affected RBC. Thus, in spite of the advantages of FLAER assay it can not totally replace the need for immunophenotyping.

Haemolytic hiatus and transfusion requirement

  1. Top of page
  2. Abstract
  3. FLAER
  4. Haemolytic hiatus and transfusion requirement
  5. Diagnosis of PNH
  6. Bone marrow analysis
  7. Classification of PNH
  8. External quality assurance program diagnosis of PNH
  9. Conclusion
  10. References

There is a significant difference in the clone size on granulocytes and RBCs. The degree of difference in previously untransfused patients varies depending on the proportion of Type III cells and the rate of haemolysis. Majority of patients in our series (14/17) showed a difference of >50% in the clone size on granulocytes and RBCs. All these patients had significant haemolysis and transfusion requirements (M. R. Madkaikar, M. R. Gupta, F. F.Jijina and K. K. Ghosh, unpublished data from our institute). However, in few patients (3/17) this haemolytic hiatus was low (<15%). The transfusion requirement in these patients was less than 2 units per year in spite of having >80% Type III clone size on RBCs. The reason for this low degree of haemolysis in these patients is not clear. It may be because of a defective complement system; however none of these patients had recurrent bacterial infections suggestive of complement deficiency. The extent of haemolytic hiatus at presentation in previously untransfused patient may give an indication of the extent of haemolysis and transfusion requirements.

Thrombosis and clone size

Clinical studies have shown that thrombosis is one of the most important life threatening complications in PNH. Two recent studies with relatively large number of patients have shown that those who have granulocyte PNH clones in excess of 50% are at a major risk of developing thrombosis (28, 29).

Diagnosis of PNH

  1. Top of page
  2. Abstract
  3. FLAER
  4. Haemolytic hiatus and transfusion requirement
  5. Diagnosis of PNH
  6. Bone marrow analysis
  7. Classification of PNH
  8. External quality assurance program diagnosis of PNH
  9. Conclusion
  10. References

PIG-A mutations can be found in the peripheral blood of virtually all healthy individuals (30). A study on the colony forming cell (CFC) assay of aerolysin resistant colonies from PNH patients and normal individuals showed, that PNH patients had clonal PIGA mutation that involved all lineages (erythroid, myeloid, and lymphoid). In contrast, PIGA mutations from healthy controls were polyclonal (i.e., did not match each other) and did not involve lymphocytes (31). PIGA mutations that arise in healthy controls occur in cells as early as colony forming units - granulocyte, erythrocyte, monocyte, megakaryocyte, but not earlier, suggesting that these mutations are probably a function of normal differentiation and have little relevance to human disease. Unlike haematopoietic stem cells, CFC have no self-renewal capacity; hence, mutations at this level will not be propagated. Healthy controls normally have up to 0.005% non-clonal PNH-like cells; thus samples with less than 0.01% are unlikely to be clonal or clinically relevant (32). Detection of this abnormal clone of PNH should ideally be done by flowcytometry using antibodies directed against at least two GPI-AP preferably against two cell lineages (33).

Standard single-color flow cytometry is sufficiently sensitive to accurately detect 3% GPI-AP-deficient cells and can be used for diagnosis of classic PNH (34). It can be further improved using two colors and careful gating of the cell populations. FLAER as a single agent is more sensitive for detection of PNH clone compared to single color immunophenotyping. In our laboratory over last 2 yr we analyzed > 100 patients of suspected PNH using both FLAER as a single agent and CD55 and CD59 using multiparametric flowcytometry. All patients who were deficient for CD55 and CD59 were also deficient for FLAER. FLAER was found to be more sensitive and showed clear separation of the positive and negative cells. Hence although there are no clear recommendations, FLAER as a single agent can definitely be used as a screening test where PNH is clinically suspected. Deficient cases then can be further investigated to determine the clone size on RBCs using CD59 and CD55. The rare inherited disorders of CD59 deficiency and PIGM gene mutation which share some of the clinical manifestations of PNH can not be diagnosed by FLAER alone; in these cases if clinically suspected analysis using CD55 and CD59 should be performed.

In subclinical PNH for the detection and monitoring of small clones on granulocytes, high sensitivity flowcytometry using at least two antibodies against GPI-AP and gating strategy using one non-GPI-linked antibody is essential (2). However, for the same purpose combining the more sensitive FLAER with multiparameter flowcytometry would definitely be advantageous (27).These cases should be investigated at diagnosis and thereafter yearly during and after treatment, even in the absence of clinical or biochemical evidence of haemolysis. This is recommended because the PNH clone size may rapidly increase in some cases and can lead to life threatening complications like thrombosis.

Bone marrow analysis

  1. Top of page
  2. Abstract
  3. FLAER
  4. Haemolytic hiatus and transfusion requirement
  5. Diagnosis of PNH
  6. Bone marrow analysis
  7. Classification of PNH
  8. External quality assurance program diagnosis of PNH
  9. Conclusion
  10. References

Although deficiency of GPI-AP can be demonstrated on haematopoietic stem cells from bone marrow, it does not have any additional diagnostic advantage and is not recommended.

PIGA mutations

The detection of PIGA gene mutation offers final confirmation to the flowcytometric diagnosis of PNH. However being technically difficult, its utility is restricted to the research setting.

Classification of PNH

  1. Top of page
  2. Abstract
  3. FLAER
  4. Haemolytic hiatus and transfusion requirement
  5. Diagnosis of PNH
  6. Bone marrow analysis
  7. Classification of PNH
  8. External quality assurance program diagnosis of PNH
  9. Conclusion
  10. References

PNH is closely related to other marrow failure syndromes especially aplastic anemia (AA) and MDS. A significant proportion of patients progress from PNH to AA/MDS and vice versa. This leads to confusion in categorizing these patients and deciding their future management. To resolve this issue International PNH Interest Group has come up with guidelines for the diagnosis and management of these patients (2). Once a significant abnormal clone is detected it should be correlated with clinical presentation (past/present history of AA or other bone marrow pathology), haemolytic parameters [complete blood count with reticulocyte count, serum concentration of lactate dehydrogenase, bilirubin (fractionated), and haptoglobin] and bone marrow analysis (bone marrow aspirate and cytogenetics) to categorize it into different subcategories (Table 3).

Table 3.   IPIG classification of PNH
  1. IPIG, international PNH interest group; PNH, paroxysmal nocturnal haemoglobinuria; AA, aplastic anemia; MDS, myelodysplastic syndrome.

AClassic PNH
BPNH in the setting of another specified bone marrow disorder (e.g., PNH/AA or PNH/refractory anemia-MDS)
CPNH sub clinical (PNH-sc) in the setting of another specified bone marrow disorder (e.g., PNH-sc/AA)

External quality assurance program diagnosis of PNH

  1. Top of page
  2. Abstract
  3. FLAER
  4. Haemolytic hiatus and transfusion requirement
  5. Diagnosis of PNH
  6. Bone marrow analysis
  7. Classification of PNH
  8. External quality assurance program diagnosis of PNH
  9. Conclusion
  10. References

There are currently two schemes available: (1) The College of American Pathologists scheme in North America that assesses red cell analysis only; and (2) The United Kingdom National External Quality Assurance Schemes (UK NEQAS-LI) for Leukocyte Immunophenotying. In a recent study by UK NEQAS, the feasibility of implementing a stabilized PNH whole-blood sample as a biological process control for PNH screening by flow cytometry was assessed (35). The study showed that PNH cells, as well as the co-existing normal red cell and granulocyte populations, remained stable for up to 120 d, both in terms of immunophenotypic and light scatter characteristics. Subsequently these samples were used for a PNH EQA program and issued to 92 laboratories worldwide. The results of this EQA program highlighted that there are significant problems faced by laboratories with both false positive and false negative results which can have serious clinical implications. These problems arise mainly because of the lack of an internationally agreed reference method for PNH clone measurement, combined with individual laboratories choice of reagent based on (un)familiarity with reactivity profiles.

Conclusion

  1. Top of page
  2. Abstract
  3. FLAER
  4. Haemolytic hiatus and transfusion requirement
  5. Diagnosis of PNH
  6. Bone marrow analysis
  7. Classification of PNH
  8. External quality assurance program diagnosis of PNH
  9. Conclusion
  10. References

Significant advances have been made in the development of new diagnostic tests and understanding of the pathobiology of PNH in the last two decades. Flowcytometry has emerged as the method of choice for screening, diagnosing, and monitoring of patients with known or suspected PNH. From the experience of our laboratory and the extensive review of the literature we have put forth the advantages and disadvantages and limitations of the various tests available for diagnosis of PNH. However, methods used for processing, combination of antibodies used for diagnosis and the gating strategies for identification of specific cell populations varies from laboratory to laboratory. Hence there is an urgent need for development of standard international guidelines for diagnosis of PNH.

References

  1. Top of page
  2. Abstract
  3. FLAER
  4. Haemolytic hiatus and transfusion requirement
  5. Diagnosis of PNH
  6. Bone marrow analysis
  7. Classification of PNH
  8. External quality assurance program diagnosis of PNH
  9. Conclusion
  10. References
  • 1
    Takeda J, Miyata T, Kawagoe K, Iida Y, Endo Y, Fujita T, Takahashi M, Kitani T, Kinoshita T. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell 1993;73:70311.
  • 2
    Parker C, Omine M, Richards S, et al. Diagnosis and management of paroxysmal nocturnal haemoglobinuria. Blood 2005;106:3699709.
  • 3
    Young NS, Abkowitz JL, Luzzatto L. New insights into the pathophysiology of acquired cytopenias. Hematology Am Soc Hematol Educ Program 2000:1838.
  • 4
    Bessler M, Hiken J. The pathophysiology of disease in patients with paroxysmal nocturnal hemoglobinuria. Hematology Am Soc Hematol Educ Program 2008:10410.
  • 5
    Endo M, Ware RE, Vreeke TM, Singh SP, Howard TA, Tomita A, Holguin MH, Parker CJ. Molecular basis of the heterogeneity of expression of glycosyl phosphatidylinositol anchored proteins in paroxysmal nocturnal hemoglobinuria. Blood 1996;87:254657.
  • 6
    Zacks MA, Garg N. Recent developments in the molecular, biochemical and functional characterization of GPI8 and the GPI-anchoring mechanism. Mol Membr Biol 2006;23:20925.
  • 7
    Rosse WF, Nishimura J. Clinical manifestations of paroxysmal nocturnal hemoglobinuria: present state and future problems. Int J Hematol 2003;77:11320.
  • 8
    Yamashina M, Ueda E, Kinoshita T, Takami T, Ojima A, Ono H, Tanaka H, Kondo N, Orii T, Okada N. Inherited complete deficiency of 20-kilodalton homologous restriction factor (CD59) as a cause of paroxysmal nocturnal hemoglobinuria. N Engl J Med 1990;323:11849.
  • 9
    Almeida AM, Murakami Y, Layton DM, et al. Hypomorphic promoter mutation in PIGM causes inherited glycosylphosphatidylinositol deficiency. Nat Med 2006;12:84651.
  • 10
    Richards SJ, Hill A, Hillmen P. Recent advances in the diagnosis, monitoring, and management of patients with paroxysmal nocturnal hemoglobinuria. Cytometry B Clin Cytom 2007;72B:2918.
  • 11
    Rosse WF. Variations in the red cells in paroxysmal nocturnal haemoglobinuria. Br J Haematol 1973;24:32742.
  • 12
    Rosse WF, Dacie JV. The role of complement in the sensitivity of the paroxysmal nocturnal hemoglobinuria red cell to immune lysis. Bibl Haematol 1965;23:118.
  • 13
    Gupta R, Pandey P, Choudhry R, Kashyap R, Mehrotra M, Naseem S, Nityanand S. A prospective comparison of four techniques for diagnosis of paroxysmal nocturnal haemoglobinuria. Int J Lab Hematol 2007;29:11926.
  • 14
    Zupanska B, Bogdanik B, Pyl H. A gel microtyping system for diagnosis of paroxysmal nocturnal hemoglobinuria. Immunohematology 2002;18:912.
  • 15
    Kinoshita T, Medof ME, Silber R, Nussenzweig V. Distribution of decay-accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria. J Exp Med 1985;162:7592.
  • 16
    Nicholson-Weller A, Spicer DB, Austen KF. Deficiency of the complement regulatory protein, “decay-accelerating factor,” on membranes of granulocytes, monocytes, and platelets in paroxysmal nocturnal hemoglobinuria. N Engl J Med 1985;312:10917.
  • 17
    Hernández-Campo PM, Almeida J, Sánchez ML, Malvezzi M, Orfao A. Normal patterns of expression of glycosylphosphatidylinositol-anchored proteins on different subsets of peripheral blood cells: a frame of reference for the diagnosis of paroxysmal nocturnal hemoglobinuria. Cytometry B Clin Cytom 2006;70B:7181.
  • 18
    Hernández-Campo PM, Almeida J, Matarraz S, De Santiago M, Sánchez ML, Orfao A. Quantitative analysis of the expression of glycosylphosphatidylinositol-anchored proteins during the maturation of different hematopoietic cell compartments of normal bone marrow. Cytometry B Clin Cytom 2007;72:3442.
  • 19
    Richards SJ, Rawstron AC, Hillmen P. Application of flow cytometry to the diagnosis of paroxysmal nocturnal hemoglobinuria. Cytometry 2000;42:22333.
  • 20
    Ware RE, Rosse WF, Hall SE. Immunophenotypic analysis of reticulocytes in paroxysmal nocturnal hemoglobinuria. Blood 1995;86:15869.
  • 21
    Hernández-Campo PM, Martín-Ayuso M, Almeida J, López A, Orfao A. Comparative analysis of different flow cytometry-based immunophenotypic methods for the analysis of CD59 and CD55 expression on major peripheral blood cell subsets. Cytometry B Clin Cytom 2002;50:191201.
  • 22
    Wang SA, Pozdnyakova O, Jorgensen JL, Medeiros LJ, Stachurski D, Anderson M, Raza A, Woda BA. Detection of paroxysmal nocturnal hemoglobinuria clones in patients with myelodysplastic syndromes and related bone marrow diseases, with emphasis on diagnostic pitfalls and caveats. Haematologica 2009;94:2937.
  • 23
    Brodsky RA, Mukhina GL, Li S, et al. Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin. Am J Clin Pathol 2000;114:45966.
  • 24
    Howard SP, Buckley JT. Activation of the hole-forming toxin aerolysinby extracellular processing. J Bacteriol 1985;163:33640.
  • 25
    Brodsky RA, Mukhina GL, Nelson KL, Lawrence TS, Jones RJ, Buckley JT. Diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) and selection of small PNH populations using a novel GPI-anchor binding toxin, aerolysin. Blood 1998;92:472A.
  • 26
    Peghini PE, Fehr J. Clinical evaluation of an aerolysin-based screening test for paroxysmal nocturnal haemoglobinuria. Cytometry B Clin Cytom 2005;67:138.
  • 27
    Sutherland DR, Kuek N, Davidson J, Barth D, Chang H, Yeo E, Bamford S, Chin-Yee I, Keeney M. Diagnosing PNH with FLAER and multiparameter flow cytometry. Cytometry B Clin Cytom 2007;72:16777.
  • 28
    Moyo VM, Mukhina GL, Garrett ES, Brodsky RA. Natural history of paroxysmal nocturnal haemoglobinuria using modern diagnostic assays. Br J Haematol 2004;126:1338.
  • 29
    De Latour RP, Mary JY, Salanoubat C, et al. Paroxysmal nocturnal hemoglobinuria: natural history of disease subcategories. Blood 2008;112:3099106.
  • 30
    Araten DJ, Nafa K, Pakdeesuwan K, Luzzatto L. Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria genotype and phenotype are present in normal individuals. Proc Natl Acad Sci U S A 1999;96:520914.
  • 31
    Hu R, Mukhina GL, Piantadosi S, Barber JP, Jones RJ, Brodsky RA. PIG-A mutations in normal hematopoiesis. Blood 2005;105:384854.
  • 32
    Brodsky RA. New insights into paroxysmal nocturnal hemoglobinuria. Hematology Am Soc Hematol Educ Program 2006;8:516.
  • 33
    Brodsky RA. Advances in the diagnosis and therapy of paroxysmal nocturnal hemoglobinuria. Blood Rev 2008;22:6574.
  • 34
    Hall SE, Rosse WF. The use of monoclonal antibodies and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria. Blood 1996;87:533240.
  • 35
    Richards SJ, Whitby L, Cullen MJ, Dickinson AJ, Granger V, Reilly JT, Hillman P, Barnett D. Development and evaluation of a stabilized whole-blood preparation as a process control material for screening of paroxysmal nocturnal hemoglobinuria by flow cytometry. Cytometry B Clin Cytom 2009;76B:4755.