Flow cytometry of GPI-AP is considered to be the gold standard of PNH diagnosis. CD55 and CD59 expression on erythrocytes are most frequently used for screening of PNH [6
]. Our presented data show that addition of further antibodies against GPI-linked antigens on different cell lineages considerably improves the sensitivity and validity of the method. Among the markers evaluated, we recommend a panel including at least the following markers: CD58 and CD59 for reticulocytes and erythrocytes a combination of CD24/66b and eventually FLAER on the granulocytes and CD14 on monocytes.
It has been shown that detection of GPI-deficient reticulocytes is more sensitive than analysis of erythrocytes, and reticulocytes better correlates with proportion of GPI-deficient white cells. The remarkable worse correlation between erythrocytes and granulocytes compared to the correlation of reticulocytes and granulocytes (Fig. ) and the larger GPI-deficient populations in reticulocyte than erythrocyte (Fig. ) are probably caused by RBC transfusions, hemolytic crises, and a shorter life span of PNH erythrocytes [24
]. As reticulocytes are less affected by these events, their examination for GPI-AP deficiency is especially suitable for diagnosis of involvement of the red cell lineage if there are small GPI-deficient granulocyte populations below 10%. Nevertheless, the most international recommendations for flow cytometry still favour erythrocyte analysis. Our data emphasise the importance of reticulocyte analysis. Even if reticulocyte clone size correlates well with white cell clone size, both should be tested to confirm the flow cytometric diagnosis of PNH because alteration of GPI-AP on leukocytes can be a sign of granulocytic/monocytic dysplasia and immaturity ([30
], data supplements) in the context of various myeloid diseases or cytolysis. Due to our results, participation of leukocytes is detected best by examination of CD14-deficiency on monocytes (Fig. ). Therefore, additional examination of monocytes provides a higher detection sensitivity of GPI-AP deficiency especially in patients with small GPI-deficient granulocyte populations <10%. In fact, the marker combination CD24/66b and CD16 seems to be suitable in sensitivity and validity to detect GPI-deficient granulocytes in our analysis.
Follow-up examination is recommended by the International PNH Interest Group (IPIG) in case of bone marrow failure syndromes [21
] even in case of initially normal flow cytometric analysis. Moreover, regular follow-up studies are indicated in case of known PNH to detect substantial changes in GPI-AP-deficient population size or cell lineage involvement, as both situations may prompt therapeutic consequences or may demonstrate effect of therapeutic interventions. Therefore, we evaluated our panel on different cell lineages for longitudinal monitoring of GPI-deficient populations to determine the frequency of significant changes in cell lineage involvement and GPI-deficient population size. Our definition of a relevant change in GPI-deficient population size (</≥50% GPI deficiency on granulocytes) is based on data published by Hall et al. [15
], demonstrating that patients with a GPI-deficient population on granulocytes ≥50% have a significant higher risk of thromboembolic events. Our results again support the importance of GPI-AP measurement on reticulocytes to obtain results independent of hemolytic crisis and RBC transfusions.
The described flow cytometry panel could be used for serial monitoring during follow-up to detect evolution of GPI-AP deficient populations and to assess therapy effects. Although follow-up investigations were performed only in a minority of patients with initially normal results (80 out of 627 patients), in seven of the follow-up patients, PNH was newly diagnosed. Based on our follow-up data with a relevant proportion of newly developing GPI-AP-deficient populations and significant changes in the size of GPI-AP-deficient populations, repeated GPI-AP analysis in regular intervals should be performed, like it is recommended by the International PNH Interest Group (IPIG) [21
]. Another interesting fact in our follow-up data is the absence of spontaneous remissions of PNH typical GPI-AP-deficient populations which had been reported. A study with 35 patients referred a rate of 15% spontaneous remissions [6
] in PNH patients, but available GPI-AP flow cytometric results and even HAM-test were limited in this study. All observed remissions in our patient group happened in the context of stem cell transplantation or intensified immunosuppressive therapy especially in patients with AA-PNH syndrome. The reported three patients with classical PNH and decrease of the GPI-AP deficient population on granulocytes below 50% developed an increase over 50% GPI-deficient cells on the granulocytes during further follow-up.
Generally, expression of GPI-anchored proteins should be analysed in all situations suspicious of PNH and in bone marrow failure syndromes [20
]. According to our data, measurement of at least two different GPI-anchored proteins on granulocytes, erythrocytes, and reticulocytes provides a simple and rapid method to detect even small GPI-deficient populations. Measurement of erythrocytes only includes the pitfall of false-negative results or false low values of GPI-deficient populations. On the contrary, examination of GPI deficiency on reticulocytes additionally prevents false positive interpretation of significant GPI-deficient populations on the leukocytes (data supplements). GPI-AP deficiencies without clinical PNH were reported in the context of other hematologic diseases or after immunosuppressive therapy [26
] as well as in healthy individuals [27
]. Our data demonstrate that in patients with small GPI-deficient populations, these can in particular be detected on monocytes. In conclusion, we recommend a flow cytometric screening panel with the markers CD58 and CD59 on reticulocytes and erythrocytes as well as with the markers CD24/66b and CD16 on granulocytes for initial diagnosis and monitoring during follow-up. In case of significant GPI-AP-deficient population in a minimum of one cell lineage, the analysis should be extended to the markers CD14 and CD48 on monocytes. Furthermore, we advise a serial monitoring for PNH patients as well as for patients with bone marrow failure syndromes without a present GPI-AP deficiency every 6 months or in case of significant clinical symptoms.
Recently, Richards et al. [29
] showed that a relevant proportion of laboratories doing PNH testing by flow cytometry have significant problems with regard to false-positive and false-negative results. On the other hand, due to the advent of a new targeted therapy option, early diagnosis and serial monitoring of PNH is gaining importance for a better patient management [17
]. As a consequence, there is the urgent need for optimised flow cytometric protocols in PNH diagnosis. In conclusion, this analysis has demonstrated that the described flow cytometric method offers significant benefits in sensitivity and validity in PNH testing and can be therefore recommended for a wider use.