Diagnosis in hematology is today more complex than in the past: in fact, it requires a number of sophisticated techniques and innovative technologies innovations that must be mutually integrated into a progressively more articulated diagnostic pathway (McKenna RW, 2000)PubMed. When a hematologic malignant disorder is suspected, diagnostic investigations should be carried out following a rational order of priority, with the aim of optimizing time and resources, as well as satisfying the diagnostic steps necessary to classify the patient according to the most recent international guidelines, such as those currently proposed by the 2008 WHO Classification (Swerdlow SH et al, 2008). Moreover, accurate biological information for each specific diagnostic context is presently needed to stratify the patient in terms of prognosis: this necessity makes the pathway even more articulated and complex, because not all diagnostic techniques are harmonized between different laboratories, nor are they readily available in all centers. The scope of this chapter is to describe a convenient diagnostic sequence that ensures efficiency for the benefit of patients and clinicians. In the remaining chapters, dedicated to specific groups of diseases, further investigations, necessary to identify for each patient specific biological characteristics that can make him suitable for targeted, will be described in detail


Examination of peripheral blood

Complete blood cell count (CBC)

In most cases, the first approach to the patient’s diagnosis is represented by the analysis of peripheral blood. This is today performed by dedicated the last generation hematology analyzers which, regardless of the technology they use, are capable of providing a fully automated CBC with differential leucocyte count in less than a minute. The evolution of automatic methods for blood cells analysis, started less than half a century ago, has continuously improved, radically transforming the operational reality of the hematology laboratory. Techniques originally developed to count particles in a fluid have been refined and extended throughout the years, so that it is currently possible to measure both size and internal characteristics of the different cell types, and to calculate mean parameters, histograms of distribution and multidimensional cytograms, based on different chemical and physical properties, which allow signal classification into distinct cellular populations. The classical CBC has turned today into a comprehensive blood profile, that includes direct quantitative measurements as well as qualitative indices, which inform about properties of red blood cells (RBCs), platelets (PLTs) and white blood cells (WBCs). New instrumental parameters are also available for all the three blood cell lines: they are useful for evaluating the cell properties and/or pointing out abnormalities with the aid of specific alarms, as to realize a true pre- microscopic guidance. Figures 1-4 shows the complete reports of normal peripheral blood CBCs obtained with four different technologies (Abbott Cell-Dyn Sapphire, Beckman Coulter LH Series, Siemens ADVIA 2120 and Sysmex XE-2100).

When CBC parameters fall within the normal range for age, sex and ethnicity (Dacie and Lewis, 2006), in the absence of specific clinical requests, the result is validated without further diagnostic investigations. Table 1 shows reference CBC ​​hematology values for healthy adults and children between the ages of 6 and 12, according to data from literature.



Table 1A. Reference values for the peripheral blood cell count in adults and 6 to 12-year-old children, according to literature data


Peripheral blood smear

In the presence of quantitative and/or qualitative alterations of the CBC, as well as when instrumental alarms are reported, a peripheral blood smear should be prepared, fixed, stained with a panoptic method and examined at the light microscope. Preparation, staining and microscopic observation of the peripheral blood smear (Figure 5-8) must accurately follow rules and standards, such as those published as international guidelines (ICSH, 1984)PubMed. Microscopic examination, in particular, should be carried out according to a systematic pattern: it should begin with the inspection of the whole smear at low magnification (10x), with the aim of assessing both quality of smear and distribution of cells, as well as the possible agglutination of PLTs (Figure 9), leukocytes, leucocytes and PLTs (Figure 10) or RBCs (Figure 11), or the formation of erythrocyte rouleaux (Figure 12). The presence of pathological cells at low frequency, that may escape the differential count procedure owing to their localization on the edges or the tail of the smear, must be carefully sought for in this phase. In addition, the importance of the observation a protein matrix on the smear background (Figure 12), that is not rarely associated with alterations of the instrumental report, should not be underestimated.


Zini_piattaforme_di-diagnosi_in ematologia_Figura_1 Zini_piattaforme_di-diagnosi_in ematologia_Figura_2 Zini_piattaforme_di-diagnosi_in ematologia_Figura_3

Fig.1A                                                          Fig.2A                                                Fig.3A

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Fig.4A                                                         Fig.5A                                                  Fig.6A

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Fig.7A                                                          Fig.8A                                                 Fig.9A

Zini_piattaforme_di-diagnosi_in ematologia_Figura_10 Zini_piattaforme_di-diagnosi_in ematologia_Figura_11 Zini_piattaforme_di-diagnosi_in ematologia_Figura_12

Fig.10A                                                       Fig.11A                                                Fig. 12A

Figure 1. The Abbott Cell-Dyn Sapphire CBC and WBC Diff report with cell distribution histograms and scattergrams – Figure 2. A Beckman-Coulter LH Series blood cell counter CBC and WBC Diff report with cell distribution histograms and scattergrams – Figure 3. Siemens ADVIA 120 CBC and WBC Diff report with cell distribution histograms and scattergrams – Figure 4. Sysmex XE-2100 blood cell counter CBC and WBC Diff report with cell distribution histograms and scattergrams – Figure 5. Peripheral blood smear: neutrophil and eosinophil – Figure 6. Peripheral blood smear: a lymphocyte – Figure 7. Peripheral blood smear: a monocyte. On the right, two platelets – Figure 8. Peripheral blood smear: a basophil granulocyte – Figure 9. Peripheral blood smear: platelet clump (in vitro artifact) – Figure 10. Peripheral blood smear: neutrophil-platelet clump (in vitro artifact) – Figure 11. Peripheral blood smear: autoimmune hemolytic anemia in a patient with lymphoproliferative syndrome at diagnosis. Massive red blood cell agglutination is visible all over the slide – Figure 12. Peripheral blood smear: rouleaux formation by stacked red blood cell. On the background, small arches formed by protein precipitation.


Microscope smear observation should then continue with a high-magnification lens (50x and/or 100x), to evaluate morphological details of all cell lines and to perform the differential WBC count. The finding of morphological alterations in the erythroid series is often the first step of a systematic diagnostic pathway: the presence of intracellular parasites (Figure 13) can lead to a diagnosis of malaria, the finding of schistocytes (Figure 14) to the diagnosis of thrombotic microangiopathy, the observation of spherocytes (Figure 15) or ellissocytes (Figure 16) to the identification of congenital hemolytic anemias; sickle cells and dacriocytes are important clues, respectively, for the diagnosis of sickle cell anemia (Figure 17) and for the suspect of bone marrow fibrosis (Figure 18). These are just a few examples of morphological characteristics that can lead to diagnoses od specific blood disorders (Bessis M, 1972Bessis M, 1076). Similarly,the possible presence of morphological changes of PLTs must be carefully searched for: when a low PLT count has been reported, the absence of pseudothrombobytopenia due to PLT aggregates or agglutinates on the smear must be confirmed (Figure 9); in the presence of true thrombocytopenia with giant PLTs the possible association with leukocyte inclusions and congenital familial disorders should be confirmed or excluded (Figure 19). The presence of circulating micromegakariocytesi or megakaryocyte naked nuclei must always be reported, since it is indicative of the presence of primary or secondary bone marrow damage.

As a routine test, the WBC differential count is usually carried out on 100 nucleated cells; in the context of onco-hematological diagnosis, on the other hand, it must be performed on a minimum number of 200 cells. The purpose of such a requirement is to increase both accuracy and precision of the count as much as possible when a hematopoietic disorder is suspected; its statistical justification can be found in the coefficient of variation of the expected result of a cell count, which is a function of the number of counted events, as shown in tables of statistical distribution (Table 2) (Rümke C L, 1977).



Table 2A. 95% confidence limits for an expected value for a cell population (“a” column) as a function of the number of cell that are counted (“n” columns). It is evident that, especially for low-frequency elements, precision and accuracy do improve as the number of total cells counted increases.


Quantitative abnormalities of leukocytes, usually well recognizable in the reports generated by all automated analyzers, may affect one or more cell lines and may appear as values ​​in excess (cytosis) and/or in defect (cytopenias). Qualitative alterations, almost always flagged by analyzers’ WBC alarms, must be carefully screened by optical microscope in all cell lines. In the presence of blasts, it is always necessary to perform a cytochemical staining for myeloperoxidase to identify positive cells as belonging to the granulocytic lineage (Figure 20); blasts that are myeloperoxidase-negative at the optical microscope (Figure 21) may still reveal a granulocytic origin when they are analyzed with other methods for the study of their immunophenotype. Cytochemical stain for non-specific esterases (Figure 22) is useful to identify blasts belonging to the monocytic line. Metachromatic stain with toluidine blue identifies basophilic granules (Figure 23). On the basis of morphological and cytochemical characteristics, it is possible to classify the majority of abnormal cells that circulate in the peripheral blood. Pathological cells that are undifferentiated from both the morphological and the cytochemical standpoint can be characterized only with the determination of the immunophenotype.

The evaluation of the peripheral blood in both qualitative and quantitative terms does play a key role in hematologic patients at the time of diagnosis, but also in all subsequent stages of their diseases, such as treatment and longitudinal monitoring.


Zini_piattaforme_di-diagnosi_in ematologia_Figura_13 Zini_piattaforme_di-diagnosi_in ematologia_Figura_14 Zini_piattaforme_di-diagnosi_in ematologia_Figura_15

Fig.13A                                                          Fig.14A                                     Fig.15A

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Fig.16A                                                         Fig.17A                                              Fig.18A

Zini_piattaforme_di-diagnosi_in ematologia_Figura_19 Zini_piattaforme_di-diagnosi_in ematologia_Figura_20 Zini_piattaforme_di-diagnosi_in ematologia_Figura_21

Fig.19A                                                         Fig.20A                                             Fig.21A

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Fig.22A                                                         Fig.23A                                             Fig.4A


Figure 13. Peripheral blood smear: three ring forms of Plasmodium falciparum are visible in the cytoplasm of the red blood cell in the center – Figure 14. Peripheral blood smear from a patient with thrombotic thrombocytopenic purpura: three schistocytes and occasional microspherocytes are visible – Figure 15. Peripheral blood smear from a patient with hereditary spherocytosis – Figure 16. Peripheral blood smear from a patient with hereditary ellissocytosis – Figure 17. Peripheral blood smear from a patient with sickle cell anemia: two drepanocytes, with crescent-like shape, are visible – Figure 18. Peripheral blood smear in a case of primary myelofibrosis (PMF), with several dacrocytes – Figure 19. Peripheral blood smear in a patient with syndrome of Sebastian. One giant platelet (on the left lower part) and a neutrophil with a Döhle body in the cytoplasm (arrow) – Figure 20. Peripheral blood smear of a patient with AML with differentiation: blasts are cytochemically posivive for myeloperoxidase (left, MGM; right, MPX) – Figure 21. Peripheral blood smear in a case of poorly differentiated AML: on the right, blasts are MPO-negative, while a eosinophil granulocyte shows the typical intense positivity – Figure 22. Peripheral blood smear in a patient with AML, monoblastic: blast cells, on the right, show a positive reaction for non-specific esterases (left, MGM; right, alpha-naphtil acetate esterase) – Figure 23. Mast cell (left, MGM; right, toluidine blue) – Figure 24. Effect on bone marrow nucleated cell counts of increasing dilution by peripheral blood with different volumes of aspirated fluid (Dresch et al, 1974).


Fine needle bone marrow aspiration biopsy

Bone marrow needle aspiration is indicated in almost all hematological malignancies and it is generally performed on the basis of an altered and/or frankly pathological peripheral blood cell count. The International Council for Standardization in Hematology ( ICSH ) has published in 2008 standardized guidelines for bone marrow examination (Table 3) and reporting (Lee SH, 2008)PubMed, in agreement with the 2008 WHO guidelines.



Table 3A. Indication for bone marrow examination, according to ICSH Guidelines (Lee et al, 2008).


From a technical standpoing, a crucial aspect is the volume of bone marrow fluid used to prepare the smears: only the first 0.2-0.5 ml of sample aspirated from the bone marrow cavity are truly representative of the actual distribution of bone marrow cells. With larger amount of fluid, contamination and dilution with peripheral blood prevents the calculation of a representative myelogram, as it is clearly described in a study published by Dresch et al in 1974 (Dresch C, 1974PubMed (Figure 24). Bone marrow drops are usually spreaded on a sufficient number of slides to obtain adequate smears; alternativel, marrow bone particles can be used to make crush preparations. The first method allows a better assessment of the marrow cellularity, megakaryocyte number, presence of low-frequency cells and/or any focal cell cluster. Cellular details, however, are not easily appreciated in large portions of the smear. Bone marrow smears, once dried, should be stained using standardized panoptic dyes. In addition, some slides should always be fixed in formalin to make possible additional specific cytochemical stainings, whenever necessary.

The microscope observation of the aspirate smears should begin under low magnification (10x and 20x objectives), starting from the feathered edge or tail, that contains marrow particles: this first step is necessary for the evaluation of cellularity, megakaryocytes (Figure 26), cell clusters of any origin and, possibly, low-frequency cells (Figures 27 and 28). At least two smears should be observed for any patient, to reduce statistical errors due to unequal cell distribution and subjectivity.

Myelogram, that is the differential cell count on aspirated bone marrow, must be performed under high magnification ( (40x, 50x, 100x objectives): in particular, a 100x objective is necessary for the best qualitative evaluation and for the identification of cellular details. When the spread marrow material is not adequate, owing to poor cellularity, peripheral blood dilution or technical errors, only a qualitative and descriptive evaluation can be possible: a myelogram in such cases would in fact not be representative of the true bone marrow cellular composition. Figure 29 shows a decision algorithm for a systematic approach to the aspirated marrow smear examination, that takes into account the variable composition and quality the of smears (d’Onofrio G, 1988). It is recommended that 500 nucleated cells be counted and classified. The myelogram should include blasts, promonocytes, promyelocytes, myelocytes, metamyelocytes, band and segmented neutrophils, monocytes, eosinophils, basophils, mast cells, erythroid precursors, lymphocytes and plasma cells (Figures 30-34). Megakaryocytes (Figure 35) and stromal cells (Figures 36-39) should be excluded from the myelogram. It is important, besides counting cells, to consider any possible abnormal finding, such as the presence of amastigotes of Leishmania donovani, that can be observed within the macrophage cytoplasm (Figure 40), as well as in free extra-cellular forms (Figure 41).


Zini_piattaforme_di-diagnosi_in ematologia_Figura_25 Zini_piattaforme_di-diagnosi_in ematologia_Figura_26 Zini_piattaforme_di-diagnosi_in ematologia_Figura_27

Fig.25A                                                         Fig.26A                                             Fig.27A

Zini_piattaforme_di-diagnosi_in ematologia_Figura_28 Zini_piattaforme_di-diagnosi_in ematologia_Figura_29 Zini_piattaforme_di-diagnosi_in ematologia_Figura_30

Fig.28A                                                         Fig.29A                                             Fig.30A

Zini_piattaforme_di-diagnosi_in ematologia_Figura_31 Zini_piattaforme_di-diagnosi_in ematologia_Figura_32 Zini_piattaforme_di-diagnosi_in ematologia_Figura_33

Fig.31A                                                         Fig.32A                                             Fig.33A

Zini_piattaforme_di-diagnosi_in ematologia_Figura_34 Zini_piattaforme_di-diagnosi_in ematologia_Figura_35 Zini_piattaforme_di-diagnosi_in ematologia_Figura_36

Fig.34A                                                         Fig.35A                                             Fig.36A

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Fig.37A                                                         Fig.38A                                             Fig.39A

Zini_piattaforme_di-diagnosi_in ematologia_Figura_40 Zini_piattaforme_di-diagnosi_in ematologia_Figura_41 Zini_piattaforme_di-diagnosi_in ematologia_Figura_42

Fig.40A                                                         Fig.41A                                             Fig.42A

Zini_piattaforme_di-diagnosi_in ematologia_Figura_43 Zini_piattaforme_di-diagnosi_in ematologia_Figura_44 Zini_piattaforme_di-diagnosi_in ematologia_Figura_45

Fig.43A                                                         Fig.44A                                             Fig.45A


Figure 25. Bone marrow needle aspirate smear in a healthy adult: the observation of several particle with low magnification permits a semiquantitative evaluation of marrow cellularity – Figure 26. Bone marrow needle aspirate smear in a healthy adult: at low magnification, megakaryocyte lineage features can be assessed in terms of quantity, quality and distribution; in this field, two normal mature megakaryocytes are visible – Figure 27. Bone marrow needle aspirate smear in a woman with metastatic breast carcinoma: a cluster of cells of non-hematopoietic origin is seen near the center – Figure 28. Non leggibile – Figure 29. Decision algorythm based on a systematic approach to the evaluatio of bone marrow needle aspirates: the different characteristics of the smeared marrow fluid are initially considered (d’Onofrio and Zini, 1996) – Figure 30. Bone marrow needle aspirate smear in a healthy adult: different cells to be included in the myelogram (a segmented neutrophil, a myeloblast and a lymphocyte) – Figure 31. Bone marrow needle aspirate smear in a healthy adult: all the cells of the granulocytic, monocytic, erythroid and lymphoid lineage must be counted to obtain the myelogram – Figure 32. Bone marrow needle aspirate smear in a healthy adult: all the cells of the granulocytic, monocytic, erythroid and lymphoid lineage must be counted to obtain the myelogram – Figure 33. Bone marrow needle aspirate smear in a healthy adult: all the cells of the granulocytic, monocytic, erythroid and lymphoid lineage must be counted to obtain the myelogram – Figure 34. Bone marrow needle aspirate smear in a healthy adult: all the cells of the granulocytic, monocytic, erythroid and lymphoid lineage must be counted to obtain the myelogram – Figure 35. Bone marrow needle aspirate smear in a healthy adult: megakaryocyte – Figure 36. Bone marrow needle aspirate smear in a healthy adult: macrophage – Figure 37. Bone marrow needle aspirate smear in a healthy adult: reticular cell – Figure 38. Bone marrow needle aspirate smear in a healthy adult: osteoblast – Figure 39. Bone marrow needle aspirate smear in a healthy adult: osteoclast – Figure 40. Bone marrow needle aspirate smear from a subject with Leishmania donovani infestation: a single extra-cellular amastigote – Figure 41. Bone marrow needle aspirate smear from a subject with Leishmania donovani infestation: two amastigotes in the cytoplasm of a macrophage – Figure 42. Bone marrow needle aspirate smear from a healthy adult: Perls’s reaction (10x) stains in blue the stromal iron stores in a marrow particle – Figure 43. Bone marrow needle aspirate smear from a healthy adult: Perls’s reaction (100x) stains in blue the stromal iron stores in a marrow particle – Figure 44. Bone marrow needle aspirate smear from an adult patient with increased iron stores (Perls’s reaction, 100x): siderocytes (above) and a sideroblast (below) – Figure 45. Bone marrow needle aspirate smear from a patient with AML with the recurrent cytogenetic aberration t(8;21) (q22;q22): in a blast cytoplasm a single thin Auer rod is present.


Bone marrow stores of iron, siderocytes and sideroblasts can be identified cytochemically, using the Prussian blue stain (Perls’s test) (Figure 42-44).

The 2008 WHO Classification of tumours of the hematopoietic and lymphoid dissues (Swerdlow SH et al, 2008) provides detailed technical and morphological criteria, both quantitative and qualitative, for the diagnosis of myeloid neoplasms.The percentage of blasts in the peripheral blood and in the bone marrow still remains the diagnostic cornerstone, not only for the first diagnosis, but also in patients’ follow-up and for the evaluation of disease progression. Flow-cytometric determination of blast cells by immunophenotype is not considered as a valide substitute for the microscope blast cell count. Histochemical stains of trephine biopsy section is however a useful aid to identify CD34+ cells in patients with hypoplastic or fibrotic bone marrow. The threshold of 20% blasts is used to discriminate myelodysplastic syndromes (MDS), with less than 20% blasts cells, and acute myeloid leukemias (AML), in which blast cells are 20% or more. As exceptions to this general rule, AMLs with specific and recurrent cytogenetical aberrations can be diagnosed even with blast percentage lower than 20%: this is the case of AML with t(8;21)(q22;q22) (Figure 45), AML with inv(16)(p13.1;q22) or (16;16)(p13.1;q22) (Figure 46) and acute promyelocytic leukmia (APL) with t(15;17)(q22;q12) (Figure 47).

According to the WHO Classification, blast cell count includes myeloblasts (with agranular or variably granular cytoplasm) (Figure 51), monoblasts (Figure 49) and megakaryoblasts (Figure 50). Erythroblasts are not included in the blast cell count, except in rare patients with the “pure” forme of AML with erythroid differentiation (Figure 51). Promonocytes (Figure 52) are considered as blasts (“blast-equivalent”) in the diagnostic context of acute AML with monoblastic or myelomonocytic differentiation. Small dysplastic megakaryoctes (Figure 53) and micromegakaryocytes (Figure 54) are not counted as blasts. In APL, abnormal promyelocytes (Figure 47) are included in the blast cell percentage.

For the diagnosis of MDS, the WHO Classification specifically requires that at least 10% of the cells in any hematopoietic lineage manifest morphologically unequivocal aspects of dysplasia to define such lineage as dysplastic. On the other hand, for the diagnosis of AML with dysplastic alterations the percentage of cells morphologically classified as dysplastic must reach a threshold value of at least 50% in any cell lineage. Morphologic alterations that define a cell as dyserythropoietic are nuclear budding (Figure 55), internuclear bridges (Figure 56), karyorrhexis (Figure 57), multinuclearity (Figure 58), multilobated nuclei (Figure 59), megaloblastic changes (Figure 60), cytoplasmic vacuolizatin (Figure 61), ring sideroblasts (Figure 62), abnormal PAS-positivity in erythroblasts (Figure 63). As for the granulocytic series, morphological aberrations that define dysgranulopoietic cells are variability of cell size (Figure 64), nuclear hyposegmentation with pseudo-Pelger nuclear morphology (Figure 65), nuclear hypersegmentation (Figure 66), abnormal, pseudo-Chediak-Higashi granulations, cytoplasmic hypgranularity or total absence of granules (Figure 68), presence of Auer rods (Figure 69). Morphologic alterations that identify a cell of the megakaryocyte lineage as dysplastic are a typical micromegakaryocyte aspect (Figure 54), nuclear hypolobularity (Figure 70) and the presence of multiple separated nuclei (Figure 71).


Zini_piattaforme_di-diagnosi_in ematologia_Figura_46 Zini_piattaforme_di-diagnosi_in ematologia_Figura_47 Zini_piattaforme_di-diagnosi_in ematologia_Figura_48

Fig.46A                                                         Fig.47A                                             Fig.48A

Zini_piattaforme_di-diagnosi_in ematologia_Figura_49 Zini_piattaforme_di-diagnosi_in ematologia_Figura_50 Zini_piattaforme_di-diagnosi_in ematologia_Figura_51

Fig.49A                                                         Fig.50A                                             Fig.51A

Zini_piattaforme_di-diagnosi_in ematologia_Figura_52 Zini_piattaforme_di-diagnosi_in ematologia_Figura_53 Zini_piattaforme_di-diagnosi_in ematologia_Figura_54

Fig.52A                                                         Fig.53A                                             Fig.54A

Zini_piattaforme_di-diagnosi_in ematologia_Figura_55 Zini_piattaforme_di-diagnosi_in ematologia_Figura_56 Zini_piattaforme_di-diagnosi_in ematologia_Figura_57

Fig.55A                                                         Fig.56A                                             Fig.57A

Zini_piattaforme_di-diagnosi_in ematologia_Figura_58 Zini_piattaforme_di-diagnosi_in ematologia_Figura_59 Zini_piattaforme_di-diagnosi_in ematologia_Figura_60

Fig.58A                                                         Fig.59A                                             Fig.60A

Zini_piattaforme_di-diagnosi_in ematologia_Figura_61 Zini_piattaforme_di-diagnosi_in ematologia_Figura_62 Zini_piattaforme_di-diagnosi_in ematologia_Figura_63

Fig.61A                                                         Fig.62A                                             Fig.63A

Zini_piattaforme_di-diagnosi_in ematologia_Figura_64 Zini_piattaforme_di-diagnosi_in ematologia_Figura_65 Zini_piattaforme_di-diagnosi_in ematologia_Figura_66

Fig.64A                                                         Fig.65A                                             Fig.66A

Zini_piattaforme_di-diagnosi_in ematologia_Figura_67 Zini_piattaforme_di-diagnosi_in ematologia_Figura_68 Zini_piattaforme_di-diagnosi_in ematologia_Figura_69

Fig.67A                                                         Fig.68A                                             Fig.69A

Zini_piattaforme_di-diagnosi_in ematologia_Figura_70 Zini_piattaforme_di-diagnosi_in ematologia_Figura_71

Fig.70A                                                         Fig.71A


Figure 46. Bone marrow needle aspirate smear from a patient with AML with the recurrent cytogenetic aberration inv(16) (p13.1;q22), associated with increased eosinophil component – Figure 47. Bone marrow needle aspirate smear from a patient with APL and the recurrent cytogenetic aberration t(15;17) (q22;q12): multiple numerous Auer rods are visible – Figure 48. Four leukemic blasts, agranular (the two above) and granular (the two below) – Figure 49. Monoblast – Figure 50. Two megakaryoblasts – Figure 51. Erythroid blasts in a patient with pure erythroid leukemia (Di Guglielmo’s disease) – Figure 52. Three promonocytes – Figure 53. Two small dysplastic megakaryocytes – Figure 54. Three micromegakaryocytes – Figure 55. Dyserythropoiesis: nuclear budding. – Figure 56. Dyserythropoiesis: internuclear bridges. Non si vede. – Figure 57. Dyserythropoiesis: karyorrhexis – Figure 58. Dyserythropoiesis: multinuclearity. – Figure 59. Dyserythropoiesis: polylobulated nucleus – Figure 60. Dyserythropoiesis: megaloblastic changes – Figure 61. Dyserythropoiesis: cytoplasmic hemoglobinization defects and vacuoles – Figure 62. Dyserythropoiesis: ring sideroblasts – Figure 63. Dyserythropoiesis: abnormal PAS-positivity in erythroid progenitors – Figure 64. Dysgranulopoiesis: variability of cellular size – Figure 65. Dysgranulopoiesis: nuclear hyposegmentation (pseudo-Pelger) – Figure 66. Dysgranulopoiesis: nuclear hypersegmentation – Figure 67. Dysgranulopoiesis: pseudo-Chediak-Higashi abnormal granules in neutrophil cytoplasm – Figure 68. Dysgranulopoiesis: hypogranularity/absence of cytoplasmic granules – Figure 69. Dysgranulopoiesis: presence of Auer rods – Figure 70. Dysmegakaryocytopoiesis: hypolobulated nuclei – Figure 71. Dysmegakaryocytopoiesis: multiple separated nuclei.



Flow Cytometry

Flow cytometry is a process of performing measurements on cells or particles that are in fluid suspension. It is a technology that simultaneously measures and analyzes multiple characteristics of single cells or particles as they pass through a beam of light. The flow cytometer is composed of three sub-systems: fluidics, optics and electronics. The fluidics system transports particles in a fluidic stream to the laser stream for interrogation. The optics system consists of excitation and collection components; the excitation components include lasers, beam shaping prisms and lenses to route the laser beams to the sample core. When particles pass through the laser (interrogation point), they scatter light and any fluorescent molecule present on the particle fluoresce. Lenses collect scattered and fluorescent light to appropriate detectors (electronic system) that converts detected light into proportional electronic signals. A cell’s or particle’s size and refractive index (forward scatter, FSC) or internal complexity (Side Scatter, SSC) could be measured by the scattered light. FSC and SSC can be used to distinguish different cell types in human blood. With the use of monoclonal antibodies (MoAb) , distinct cell populations can be identified by the markers present on the cell surface. The flow cytometer can detect blood cells based on the presence of MoAb with bound fluorochromes. A fluorochrome is a functional molecule that fluoresces or emits light when it is excited by energy source (such as a laser beam). Each fluorochrome can be characterized by its unique excitation and emission spectra, thus indicating the range of wavelengths that will excite the fluorochrome.

When we can use

Immunophenotyping has become a major diagnostic tool in addition to morphology , owing to its accuracy and speed. Cellular biomarkers detected by flow cytometry allow a proper definition of hematological malignancies ‘ lineage and differentiation. Indeed, immunophenotyping allow to distinguish the acute myeloid leukemia (AML) from the acute lymphoblastic leukemia (ALL) as well as different subtypes on the basis of differentiation within the two groups. Similar results come from the analysis of lymphoproliferative disorders (LPDs). In order to obtain a correct diagnosis of hematological malignancies the European Group for the immnunological Characterization of Leukemias (EGIL) established a panel of 27 antigens divided in a first set of few markers (the screening) for quick orientation and the complete panel to define the pathology (Benè MC et al, 2011)PubMed . Finally, the correct panel of MoAb used at diagnosis allows to follow the minimal residual disease (MRD) afterwards.

Screening in AL

A set of few MoAb is necessary to investigate the origin of cytopenias , leucocytosis as well as the lineage involved in AL. The CD45/side scatter (SSC) allows to distinguish specific populations such as granulocytes, monocytes, lymphocytes and hematopoietic precursors on the basis of their respective expressions of the marker. The immature hematopoietic cells and the blast population of the majority of AL may be found in the low SSC/low-intermediate CD45 area as defined “bermudes” area (Fig. 1)  (Matarraz S et al, 2010)PubMed. The usefulness in identifying the blast population should invite us to put this marker in each tube. Cytoplasmic and nuclear markers are relevant in the lineage assessment and the following markers are proposed in the screening analysis:cCD3, MPO, TdT, and CD79a. Cytoplasmic CD3 and CD79a are one of earliest events occurring after commitment of a progenitor cell toward the T or B lymphoid lineage, respectively. Therefore, these markers may be used to identify T- or B- ALL. Anyway , the presence of these markers in lymphomas suggests the use of surface markers CD3 and CD19 to distinguish the two entities. The terminal deoxiribonucleotidyl transferase (TdT) is a DNA-repairing enzyme solely present in the nucleus of B- and T- ALL blasts and useful to distinguish ALL from lymphomas. The MPO enzyme may be easily found in myeloid cells and expressed from the AMLs although minimally differentiated , erythroid , megacariocytic some monocytic AMLs are negative. Indeed, other myeloid markers such as CD13 and CD33 in absence with lymphoid – lineage markers (eg. CD19,CD79a, cCD3) may be used to confirm the diagnosis of AML. The co-expression of previously described lymphoid and myeloid markers suggest a diagnosis of mixed phenotype acute leukemia (Matutes E et al, 2012) (Fig. 2). Finally , the immaturity marker CD34 is often expressed by leukemic blasts and may be considered in this first panel.


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Fig.1B                                                          Fig.2B


Fig.1 Different subpopulations evidenced by CD45/SSC. Bermudes area includes stem cells, basophils, mast cells and dendritic plasmocytoid cells. Matarraz S. et al. Clin Cytometry 2010 – Fig.2 Blasts in R1 co-express B-lineage and myeloid markers: CD34/TdT/Cd79a/CD19/ MPO/CD13 (AML with mixed phenotype).   (Matutes E et al. 2011)PubMed.



Complete panel

The second set of antigens confirms the diagnostic orientation provided by the screening. During B-cell differentiation, CD22 and CD19 are first differentiation antigens appearing to cells’ surface (McKenna RW et al, 2001)PubMed. According to WHO2008, CD19 is required for B- lineage assignment together with one or two other B-lineage antigens in case of strong or dim expression of CD19, respectively. Warnings on the expression of CD19 and CD22 antigens come from the aberrant expression of the first one in AML and the expression of CD22 in AML with basophilic or mast cell differentiation. With the previous markers also the CD10 is used to confirm diagnosis of B-ALL because it is expressed in the majority of childhood ALL and in many adults ALL. Generally, the bright expression of CD10 identifies the phenotype “common” (B II) while the low expression characterizes the pre-B stage of ALL (B III) (Fig 3-4). However, the presence of cytoplasmic immunoglobulin µ-chain in absence of surface immunoglobulins adds another relevant information to the definition of the pre-B stage of ALL. The assessment of surface immunoglobulin expression contributes to distinguish Burkitt’ s lymphomas or mature ALL Burkitt-like (B IV) from the other stages of B-ALL. During T-cell differentiation, CD7 and CD2 appear early during the bone marrow maturation thus showing their usefulness in the definition of first T-ALL stages (T I and T II). Within the other early T-cell markers, the CD5 can be also considered for the identification of T-ALL . The surface CD3 represents an important antigen to differentiate the more mature stage T IV from the other stages where it is generally absent. The antigen CD1a signs the cortical stage of ALL when it is associated to CD4 and CD8 double positive antigens (T III). The immunephenotypic analysis of AML (Orfao A et al, 2004) PubMed has to comprise the HLA-DR antigen which is expressed in the majority of AML with the important exception of APL. Indeed , this marker may be used to discriminate the APL from the other types (Fig 5). The antigen CD117 has been found in more than 75% of AML thus representing a mandatory marker in this panel (Fig 6). When co-expressed with the CD64 , CD33 and CD13, the CD14 is an hallmark of monocytic and myelo-monocytic AML. It could be useful also for the separation of leukemic cells from monocytes. The CD65 and CD15 antigens appear early during the granulo-monocytic differentiation (Fig. 7)although the CD65 seems to be more specific than CD15 on the granulocytic pattern because of the possibility of the latter to be found in B-ALL. Anyway, CD15 is useful to exclude remaining mature granulocytes . CD16 as well as CD11b and CD64 can be tested to further identifies mature granulocytic and monocytic compartments , respectively. Finally, platelet integrins CD41/gpIIb or CD61/gpIIIa may be used to diagnose or exclude megacariocytic leukemias. On the other hand, the transferrin receptor (CD71), the glycophorin A (CD235) or the trombospondin receptor (CD36) may be tested to identifies erythroblastic leukemias. The panel should be include the CD56, the neural cell adhesion molecule which normally is expressed on NK lymphocytes and activated monocytes. The marker CD56 is expressed from 10% to 40% of AML , depending on the subtype. In plasmocytoid dendritic cells neoplasias the antigen CD56 represents the most relevant marker expressed, usually together with the CD4 ,the HLADR and the interleukin -3 receptor CD123 (Fig. 8). The last of them identifies also basophils which could be discriminated on the basis of the absence of HLA-DR. It is important to underline that CD123 is expressed in the subset of B-ALL CD34-. The lymphoid markers CD7 and CD4 have been described as aberrant phenotypes in the majority of AML with a frequency ranging from 20% to 40% on the basis of subtype studied.


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Fig.3B                                                          Fig.4B                                             Fig.5B

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Fig.6B                                                          Fig.7B                                             Fig.8B


Fig.3 Three subsets of B lymphocytes CD 19 positivi are identified: pop. 1 (green) CD22/CD34/TdT/CD38/CD10bright (pro-B), pop.2 (red) CD22/CD20 eter /CD10dim/CD38/TdT /CD34 (pre-B ) e pop.3 (violet) CD22/CD20/CD10/CD38 /TdT/CD34 ( mature lymphocytes). – Fig.4 Lymphoblasts (red) with the abnormal expression of CD22Hehet e CD 10bright compared to hematogones ( violet) CD22Omog/CD34 . Mc Kenna et al. Blood 2001 – Fig.5 Pattern of expression of APL CD45/CD117/HLA-DR /CD11b – – Fig.6. Phenotypic pattern of AML with minimal differentiation CD34/CD13/CD15/CD33 /MPO Fig.7. Pattern of AML M2 (according to FAB classification) with the aberrant expression of CD19 – Fig.8 Leukemic blasts show CD123/HLA-DR/CD4/CD56: dendritic neoplasia



Screening in LPDs

Screening analysis shows an important role to guide the sub-sequent steps of diagnosis when either a lymphocytosis and a lymphopenia was found. Markers as CD19, CD3 and CD56 are mandatory to investigate the lineage- B,-T and natural Killer(NK) , respectively. Then, K and lambda expression provide a clear definition of the clonality in order to differentiate early benign from pathological B- lymphocytosis (Fig 9). Markers as CD4 and CD8 are useful to evaluate the different sub-population of T-lineage lymphatic disorder.  Finally, CD45/SSC is useful for a better discrimination of pathologic plots from normal ones.

Complete Panel in LPDs

When a B-lineage clonality was evidenced other antigens will confirm the lineage specific differentiation and allow the further classification within the B-cell disorders. From markers used in LPDs, the CD5 represents the most important marker to identify the chronic lymphocytic leukemia and mantle cell lymphoma. Anyway, other combinations of MoAb are needed to differentiate the two entities and diagnose the other ones. In particular, the CD23 co-expressed with CD5 characterizes the CLL (Fig 10) (Zeppa P et al, 2004)PubMed although it is relevant to have a decreased expression of surface light chains as well as CD22, CD79b, and CD20. During the last years other markers have contributed to reach a more sure diagnosis of CLL. CD81, a tetraspanin acting in conjunction with the CD19 on normal B cells, appears down regulated in CLL while the CD43 and CD200 are up-regulated. Burkitt’s and follicular lymphomas (Fig. 11) such as B-cell precursor ALL show the markers CD10 and CD38 associated to the absence of CD5. Many cases of diffuse large B-cell lymphomas show the same features. Indeed, during the analysis of a bone marrow, it should be underlined the importance of the combination CD19/CD10/CD38/CD20 for the distinction of hematogones from pathological lymphocytes (McKenna RW et al, 2001)PubMed. Finally, CD103, CD25 and CD11c are useful to identify the hairy cell leukemia. When a T –lineage population was identified from the screening , the panel to classify the T-cell disorder should comprise antigens such as CD2, CD3,CD4,CD8,CD5 and CD7. Majority of them were investigated to observe an up- or down- regulation on the surface of T-cell population. In the presence of CD4+ cells associated to the absence of CD7 a diagnosis of Sezary Syndrome is pertinent. On the other hand, the positivity of the antigen CD10 on CD3+ cells may suggest an angioimmunoblastic T-cell lymphoma. Once investigated the type of the T-cell receptor (αβ or γδ),further investigations should be oriented to the study of the clonality by a panel of Vβ specifities of T-cell receptor (Fig 12). Within CD56+ disorders other antigens as CD57, CD16 and intracytoplasmic granzyme B or perforine may be used in the exploration of NK-disorders. Anyway, it should be also considered the hypothesis of malignant plasma cells in front of a predominant CD56+ population.

Minimal Residual Disease

Despite a high remission rate ,a significant number of patients with acute leukemia (AL) relapse. Thus, the evaluation of minimal residual disease (MRD) in AL is an important strategy to better identify high risk patients. The study of MRD in AML is more difficult than ALL due to more heterogeneous blasts in AML. Thus, monitoring of MRD in AML needs of the detection of leukemia-associated immunophenotypes (LAIPs) (Benè MC et al, 2010; Kern W et al, 2004PubMed ; Al-Mawali A et al, 2008; Campana et al, 1999Pubmed ). Different categories of LAIPs can be defined: 1) antigen over-expression , e.g. abnormally increased expression of CD33 or CD34 antigens on myeloid blasts; 2) lack of antigen expression , e.g. of HLA-DR; 3) asynchronous antigen expression means the expression of different antigens across the borders of maturation stages, e.g. the expression of early (CD34 or CD117) and late markers, such as CD11c, CD14, CD65 antigens; 4) cross-lineage antigen expression refers to aberrant expression of antigens which are normally found on other hematopoietic lineages on AML blasts , e.g. CD2, CD3, CD5, CD19 or CD56 antigens on myeloid blasts; 5) aberrant scatter pattern with respect to size and granularity (Fig 13-15). Depending on the choice of applied antibodies, the sensitivity for detection of the leukemic cells with MFC ranges in AML cases between 0.1% and 0.01% of all nucleated cells. Several studies investigated the value of MFC for MRD monitoring in larger cohorts of patients. All demonstrated strong correlations of the residual levels of leukemic cells after therapy with clinical outcome. Thresholds of 0.1% and 0.01% are generally accepted to separate MRD-positive from MRD-negative patients in AML and ALL, respectively. Within LPDs arises the importance of monitoring the MRD during the follow-up of patients, particularly in CLL. Thus a combination of MoAb by four-color flow cytometry was defined: the antigens CD5 e CD19 were associated with CD20/CD38, CD81/CD22 and CD79b/CD43 (Rawstron AC et al, 2007)PubMed.


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Fig.9B                                                          Fig.10B                                             Fig.11B

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Fig.12B                                                         Fig.13B                                             Fig.14B

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Fig.9 Screening in lymphocytosis: comparison between the normal pattern of k/λ chains (fig 11a) and the pathological pattern displaying a clonality for λ-chain – Fig.10 CLL with the co-expression of CD19/CD5/CD23 and clonality for K-chain. Zeppa P et al Cancer 2004 – Fig.11 The co-expression of both CD19 and CD10 antigens in the di bcl-2 positive population identifies a follicular lymphoma. Zeppa P et al Cancer 2004 – Fig.12 Study of clonality in a t-lineage population with an heterogeneous expression of CD5 and CD8. (Vβ-13.2/ Vβ-4 positive) – Fig.13 Leukemia –associated immunophenotype(LAIP) in AML. Al-Mawali A. et al. Am J Clin Path 2008 – Fig.14 Monitoring of MRD . Benè MC et al. Haematologica 2010 – Fig 15 Blasts gated by CD45/SSC ed SSC/FSC show the LAIP CD34/CD117/CD7. Al-Mawali A. et al. Am J Clin Path 2008.



Molecular Diagnostics in Hematology


Molecular diagnostics of blood diseases aims to identify distinctive genetic abnormalities at DNA or RNA level associated to specific nosographic entities. The new technologies of whole genome study allowed the identification of new molecular biomarkers and enriched the panel of diagnostic assays available for the hematologist.

Methods to study molecular abnormalities in hematology include the Polymerase Chain Reaction (PCR), the real time quantitative PCR (RQ-PCR), the Gene Sequencing with Sanger method, the Next Generation Sequencing (Deep Sequencing), the Comparative Genomic Hybridization (CGH), the Gene Expression Profiling with microarray technology, the Single Nucleotide Polymorphism (SNP) Array. With the exception of PCR, RQ-PCR and Gene Sequencing with Sanger methods, the other technologies do not represent routine assays in the common laboratory practice due to high costs and to technical and interpretative difficulties, but allowed the identification of new genetic lesions and the following development of easier and cheaper assays with wider use in the routine molecular diagnostics.

The Polymerase Chain Reaction (PCR) is the pivotal assay to study molecular abnormalities in hematology and is used to identify fusion gene transcripts derived from recurrent chromosomal translocations, rearrangements of genes codifying immunoglobulins and T-cell receptos, recurrent mutations in oncogenes and tumor suppressor genes, enzymatic and coagulation factor polymorphisms. The finding of a distinctive genetic lesion can have diagnostic and prognostic value, can be used as minimal residual disease marker and address the treatment choice toward a targeted therapy. PCR method utilizes the Taq polymerase enzyme and synthetic oligonucleotides (primers) to amplify DNA fragments through cycles of denaturation, annealing and elongation performed in a thermocycler. The template of the PCR reaction can be genomic DNA isolated from bone marrow (BM) mononuclear cells, peripheral blood (PB) leucocytes or tissue samples. The Reverse Trascriptase PCR (RT-PCR) amplifies cDNA obtained from RNA retrotranscription and allows the study of molecular abnormalities of expressed genes, such as fusion gene transcripts from recurrent chromosomal translocations.

Primers are 20-30 nucleotide sequences complementary to 5’ and 3’ ends of the double helix of DNA fragments to amplify, respectively. To identify fusion gene transcripts, the former oligonucleotide is complementary to the sequence belonging to one chromosome, the latter oligonucleotide to the sequence belonging to the other chromosome. The reaction will amplify only the samples carrying the chromosomal translocation target.

To identify point mutations and polymorphisms, either oligonucleotide is complementary to the mutated sequence, giving rise to an amplification product only in presence of the mutation (Allele-specific oligonucletide PCR, ASO-PCR). Alternatively, mutations or polymorphisms can be within the amplified sequence and be recognized by restriction enzymes that cut DNA where the mutation is: in this case, the presence or absence of a given mutation can introduce or delete the restriction site for the enzyme (Restriction Fragment Lenght Polymorphysm PCR, RFLP-PCR). To increase the method sensibility and specificity, the nested-PCR uses oligonucleotides matching within a primary PCR product, which works as template.

PCR amplification products are visualized by electrophoresis on agarose gels. Moreover, the Melting Curve Analysis can visualize the presence of a given mutation within the amplified product through real time thermocyclers. This method is founded on the different dissociation properties of the double DNA helix related to single nucleotide substitution visualized by fluorochromes as SYBR green o Eva green, with or without the use of labeled probes.

For mutation screening, the PCR product can be sequenced according to Sanger’s method, which utilizes modified dideoxynucleotidetriphosphate (ddNTP) lacking a hydroxilic group at carbon 2’ and 3’ of the ribose molecule. ddNTP are incorporated into the newly synthesized DNA strand by DNA polymerase with the following elongation interruption in different points before the end of duplication of the DNA template. ddNTP are labeled with radioactive isotopes or fluorescent dyes allowing the visualization of DNA strands of different length after electrophoretic separation. The traditional approach works by four different PCR reaction, one for every single ddNTP utilized, followed by electrophoresis on polyacrylamide gel. Automatic sequencers perform one single sequencing reaction with all four ddNTPs (ddGTP, ddATP, ddCTP, ddTTP) labeled with different fluorochromes, so each DNA strand gives a light emission of different color according to the terminal ddNTP. The result will be a sequence of different colored peaks corresponding to single nucleotides. Mutations and polymorphism can be identified by matching with reference sequences available on online database (GenBank, RefSeq).

The real-time or quantitative PCR allows DNA amplification and quantification by use of fluorochromes such as SYBR green, which intercalate into amplified DNA inducing a light signal as intensive as the copies of PCR product. More commonly quantitative PCR utilizes a complementary oligonucleotide (probe) matching the internal sequence of the amplified product and labeled with two fluorochromes, the former (reporter) at the 5’ end e the latter (quencher) at the 3’ end. During denaturation, annealing and elongation cycling, the probe binds the DNA template between forward e reverse primers. When Taq Polymerase, which is provided of exonuclease activity, meets it during DNA replication, the probe is degraded and the quencher is drifted apart from the reporter, which can produce a fluorescent signal proportional to the degradation of the probe and to the number of amplified PCR products. The fluorescence curve has a sigmoidal trend where the cycle at which the fluorescence starts to increase exponentially is named threshold cycle (Ct). Transcript expression value is calculated on the base of a reference gene (such as GAPDH, ABL, etc.) which is amplified contextually. A standard curve with progressive dilutions of plasmids carrying the target gene and the reference gene is used to calculate the copy numbers.

Real time PCR is commonly combined to RT-PCR (qRT-PCR) to quantify expression levels of target genes used as biomarkers to monitor the minimal residual disease (MRD).


Routine Molecular Diagnostic Assays in Hematology

Molecular diagnostic assays in oncohematology should be performed according specific indications related to clinical data and first level laboratory and morphology results. In many cases, the finding of certain molecular abnormalities identifies distinct nosographic entities with well-defined clinical and biological characteristics. In other cases, the presence of some molecular abnormalities, although not specific of a distinct disease, can influence prognosis and therapeutic choices.

Molecular abnormalities routinely searched in the diagnostic work-up of neoplasms of hematopoietic and lymphoid tissue include fusion genes deriving from recurrent chromosomal translocations, oncogene mutations and expression. The diagnostic panel is continually evolving and in the last years has been further enriched thanks to technological advancements reached with the Gene Expression Profiling and especially the Next Generation Sequencing (Deep Sequencing).

Tables 1 and 2 summarize the most common molecular abnormalities (mutations and fusion genes) used in the diagnostic work-up of acute leukemia. Table 3 shows the molecular abnormalities used in the diagnostic work-up of myeloproliferative neoplasms. Table 4 summarizes molecular assays used in the study of lymphoproliferative neoplasms. The description of the methods most commonly used in the molecular biology laboratories of Hematology Departments follows.



Table 1C: Fusion genes which identify distinct nosographic entities with recurrent genetic abnormalities among Acute Leukemias according WHO classification (2008)



Table 2C: Recurrent gene mutations in AML patients with impact on the outcome (adapted from Patel et al, 2012)



Table 3C: Common genetic abnormalities in patients with Chronic Myeloproliferative Neoplasms



Table 4C: Molecular assays used in the diagnostic work-up of Lymphoid Neoplasms




BCR-ABL fusion gene, resulting from the chromosomal translocation t(9;22)(q34;q11) (Philadelphia chromosome) is the molecular biomarker of Chronic Myeloid Leukemia (CML) and is also present in 5 % of pediatric Acute Lymphoblastic Leukemia (ALL), in 20 to 50% of adult ALL and in less than 2% of adult Acute Myeloid Leukemia (AML). It has been the first molecular abnormalities correlated to a distinct disease.

This translocation joins 3’ sequence of c-ABL proto-oncogene, codifying a tyrosine kinase, on chromosome 9 with the 5’ sequence of BCR gene on chromosome 22. Breakpoints on c-ABL occur at 5’ of exon 2 and less commonly at intron 2. Breakpoints on BCR gene are more variable and occur at intron 13 and 14 (major breakpoint cluster region, M-bcr), at exon 1 (minor breakpoint cluster region, m-bcr) or between exons 19 and 20 (micro breakpoint cluster region o µ-bcr), giving rise to different fusion transcripts. The BCR-ABL fusion protein can have a molecular weight of 190 kDa (p190), 210 kDA (p210) e 230 kDa (p230), according to the breakpoints on BCR gene. BCR-ABL p190, resulting from e1a2 (95%) or e1a3 (rare) transcripts, with the m-bcr breakpoint on BCR gene, occurs in 60% of Ph+ ALL and in sporadic cases of AML (Fig. 1).

BCR-ABL p210, resulting from b3a2 (55%), b2a2 (40%), b2a3 (rare) and b3a3 (rare) transcripts with the M-bcr breakpoint on BCR gene, occurs in almost all cases of Ph+ CML and in 40% of Ph+ ALL (Fig. 2).

BCR-ABL p230, with the µ-bcr breakpoint on BCR gene, occurs in some cases of Ph+ CML with indolent clinical course.

The finding of BCR-ABL fusion gene has high impact in the diagnostics of Chronic Myeloproliferative Neoplasms and Acute Lymphoblastic Leukemias, because further defining specific nosographyc entities with distinct clinical and prognostic characteristics, it identifies patients suitable of a target therapy with tyrosin kinase inhibitors, such as imatinib (Glivec), dasatinib (Sprycel) e nilotinib (Tasigna), which inhibit the BCR-ABL fusion protein activity and can lead to hematological, cytogenetic and molecular remission of disease.

BCR-ABL fusion transcript is routinely identified by nested-RT PCR (van Dongen JJ et al, 1999)PubMed, with two distinct reaction for p190 and p210 isoforms (Fig. 1 and Fig. 2). The sensitivity of this method is 10-4–10-5. BCR-ABL search is performed on RNA isolated from BM or PB. Patients positive for BCR-ABL are monitored by real time quantitative RT-PCR on RNA isolated from in toto PB leukocytes after osmotic lysis of red blood cells. BCR-ABL copies are calculated by standard curve obtained with progressive dilutions of the plasmid with BCR-ABL insert. BCR-ABL levels are expressed on international scale as ABL percentage and standardized through a conversion factor calculated for each laboratory. A BCR-ABL level less than 0.1% is considered as Major Molecular Response (MMR) in CML patients treated with tyrosine kinase inhibitors (Hughes T et al, 2006)PubMed. Quantitative BCR-ABL assay is performed in Ph+ CML patients every three months (Baccarani M et al, 2009)PubMed .

BCR-ABL mutation screening is indicated: 1) at diagnosis only in CML patients with accelerate and blastic phase; 2) during first-line imatinib therapy in case of failure, in case of an increase in BCR-ABL transcript levels leading to MMR loss or in any other case of suboptimal response; 3) during second-line dasatinib or nilotinib therapy in case of hematologic or cytogenetic failure. BCR-ABL mutation screening is performed through direct sequencing alone or combined with denaturing-high performance liquid chromatography (D-HPLC) analysis where available (Soverini et al, 2011)PubMed.



PML-RARA fusion gene occurs in Acute Promyelocytic Leukemias (APL) carrying the chromosomal translocation t(15;17)(q22;q21). The breakpoint on the gene RARA codifying the receptor alpha for the Retinoid Acid on chromosome 17 is located at the level of intron 2. Three regions of PML gene on chromosome 15 can be involved by breakpoints in this translocation: intron 6 (bcr1; 55%), exon 6 (bcr2; 5%), and intron 3 (bcr3; 40%).

There are two PML-RARA isoforms: the Long form (L), corresponding to bcr1 and bcr2 transcripts, and the Short form (S), corresponding to bcr3 transcripts. The two isoforms are identified by two distinct nested RT-PCR reactions (van Dongen JJ et al, 1999)PubMed (Fig. 3). The S form has a worse prognosis. The sensitivity of this method is about 10-4. Most patients treated with combination ATRA-antracycline therapy reach the molecular remission. The persistence of the PML-RARA transcript at the end of consolidation therapy is highly predictive of disease relapse. PML-RARA monitoring in the follow-up of patients with APL is routinely performed on BM samples every three months. Recurrence of RT-PCR positivity is highly predictive of relapse, with a median interval of about three months between the molecular and hematological relapse of APL. PML-RARA transcript levels can be evaluated by qRT-PCR. No relationship between PML-RARA log reduction after induction and relapse risk was observed, but sequential MRD monitoring to detect persistent or recurrent PCR positivity has been shown as the strongest independent prognostic factor for clinical relapse and relapse-free survival (Grimwade D et al, 2009)PubMed .


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Fig.1C                                                          Fig.2C                                             Fig.3C


Fig.1 Electrophoresis on agarose gel of nested RT-PCR for BCR-ABL p190. Lanes 1 e 2: negative samples; Lanes 3: positive sample for e1a2 transcript; Lane 4: positive control; Lane 5: negative sample; Lane 6: molecular weight 100 bp (Courtesy of Dr F. Guidi, Institute of Hematology, Università Cattolica del Sacro Cuore, Rome) – Fig.2 Electrophoresis on agarose gel of nested RT-PCR for BCR-ABL p210. Lanes 1-6 and 9: negative samples; Lane 7: positive control for b2a2 transcript; Lane 8: positive control for tra b3a2; Colonna 10: controllo positivo per trascritto b3a2; Colonna 11: controllo negativo; Colonna 12: pesi molecolari 100 bp (Courtesy of Dr F. Guidi, Institute of Hematology, Università Cattolica del Sacro Cuore, Rome) – Fig.3 Electrophoresis on agarose gel of nested RT-PCR for PML-RARA. Isoform L: lanes 1-8 (lane 1: positive sample; lanes 2-6: negative samples; lane 7: positive control; lane 8: negative control). Isoform S: lane 10-15 (lanes 10-13: negative samples; lane 14: positive control; lane 15: negative control). Lane 10: 100 bp molecular weight. (Courtesy of Dr F. Guidi, Institute of Hematology, Università Cattolica del Sacro Cuore, Rome).



RUNX1-RUNXT1 fusion gene (also named AML1-ETO) is the result of the translocation t(8;21)(q22;q22) which occur in 7% of de novo AML and in rare cases of therapy-related AML. According to WHO classification, AMLs with t(8;21)(q22;q22) and RUNX1-RUNXT1 fusion gene are classified among leukemias with recurrent genetic abnormalities and are commonly associated with good prognosis. RUNX1-RUNXT1 fusion transcript can be evidenced by nested RT-PCR in all AML patients with t(8;21)(q22;q22), but also in some patients without this translocation at conventional cytogenetics or with complex karyotype (van Dongen JJ et al, 1999)PubMed. PCR products are constant in sizes and results from in-frame fusion between exon 5 of RUNX1 and exon 2 of RUNXT1 (Fig. 4). The significance of RUNX1-RUNXT1 positivity during follow-up is still uncertain because it can persist at long term in patients in disease remission after allogenic transplantation. However, the quantification of RUNX1-RUNXT1 transcript by qRT-PCR on BM samples after induction and consolidation therapy and on BM and PB samples during follow-up significantly affects the relapse risk: a > 3 log reduction in RUNX1-RUNX1T1 after course 1 induction chemotherapy is associated to a significantly lower cumulative incidence of relapse (CIR), while, a 100% relapse rate was associate to a MRD threshold of > 500 copies in BM samples and > 100 copies in PB samples during follow-up (Yin JA et al, 2012)PubMed .



CBFB-MYH11 fusion gene is the result of the pericentromeric inverision inv(16)(p13;q22) or of the chromosomal translocation t(16;16)(p13;q22), following which the gene CBFB (core binding factor β subunit), localized on chromosome 16q22 is fused with the gene MYH11 (myosin heavy chain 11) on chromosome 16p13. The breakpoint of CBFB occurs at level of intron 5, while the 5’ region of MYH11 gene is deleted by the chromosomal aberration. Although ten different transcripts have been described, the transcript A justify 85% of cases. The inv(16)(p13;q22) and t(16;16)(p13;q22) are difficult to identify with conventional cytogenetics, while CBFB-MYH11 fusion transcript is commonly identified by RT-PCR (van Dongen JJ et al, 1999)PubMed; (Fig. 5).

CBFB-MYH11 fusion transcript occurs in 5-8% of all cases of AML, often with granulocytic and monocytic differentiation and abnormal eosinophil BM component. It is commonly associated to good prognosis. The significance of CBFB-MYH11 fusion transcript in the follow-up of AML patients carrying inv(16)(p13;q22) or t(16;16)(p13;q22) has given uncertain results. PCR negativity after consolidation therapy and during follow-up has been associated to lower risk of relapse. CBFB-MYH11 transcript levels evaluated by qRT-PCR are highly predictive of relapse risk: a level < 10 copies of CBFB-MYH11 per 105 copies of ABL on PB samples after induction and consolidation therapy is associated to the lowest CIR, while cut-off MRD thresholds associated with a 100% relapse rate are > 50 copies in BM and > 10 copies in PB (Yin JA et al, 2012)PubMed .


Zini_piattaforme_di_diagnosi_in_ematologia_Figura_4 Zini_piattaforme_di_diagnosi_in_ematologia_Figura_5

Fig.4C                                                          Fig.5C


Fig.4 Electrophoresis on agarose gel of nested RT-PCR for AML1-ETO. Lane 1: 100 bp molecular weight; lane 2-5: negative samples; lane 6: positive sample; lane 7: positive control; lane 8: negative control (Courtesy of Dr F. Guidi, Institute of Hematology, Università Cattolica del Sacro Cuore, Rome) – Fig.5 Electrophoresis on agarose gel of nested RT-PCR for CBFB-MYH11. Lane 1: positive sample; lane 2-5: negative samples; lane 6: positive control; lane 7: negative control; lane 8: 100 bp molecular weight. (Courtesy of Dr F. Guidi, Institute of Hematology, Università Cattolica del Sacro Cuore, Rome).


FLT3 mutations

The fms-like receptor tyrosine kinase-3 (FLT3) is a class 3 tyrosine kinase associated membrane receptor, which is mutated in about one third of AML patients. The most common mutations are the internal tandem duplications (ITD) of the sequence codifying the junxta-membrane domain and the missense mutations at aminoacid D835 of the kinase domain (KDM), which occurs in 15-35% and 5-10% of AML patients, respectively. Other mutations close to position D835 have been described, including insertion, point mutation and deletions. FLT3 mutations represent the most common genetic abnormalities described in AML patients. FLT3 ITD mutations are associated to leukocytosis and poor prognosis. The clinical significance of FLT3 KDM is still debatable.

The identification of FLT3 mutations is recommended in all AML patients because is important for risk stratification and for treatment choice. Patients with FLT3 mutations can be treated with FLT3 inhibitors in combination with conventional chemotherapy. Moreover FLT3 mutation assay can be utilized as marker of Minimal Residual Disease.

FLT3 ITD assay is performed by a RT-PCR which shows an adjunctive band on gel electrophoresis, bigger in size compared to wild type band (Fig. 6) (Nakao M et al, 1996)PubMed.

FLT3 KDM assay consists in a RT-PCR followed by digestion of amplified product with the Eco RV restriction enzyme: wild type samples are completely digested by the enzyme resulting in two bands of 69 and 46 bp respectively, while mutated samples are only partially digested with persistence of undigested 114 bp band (Fig. 7) (Yamamoto Y et al, 2001)PubMed.


Zini_piattaforme_di-diagnosi_in ematologia_Figura_6 Zini_piattaforme_di-diagnosi_in ematologia_Figura_7

Fig.6C                                                          Fig.7C


Fig.6 Electrophoresis on agarose gel of RT-PCR for FLT3 ITD. Lane 1, 6, 7 and 8: mutant samples; lanes 2: 3, 4 and 5: wild type samples; lane 9: negative control (Courtesy of Dr F. Guidi, Institute of Hematology, Università Cattolica del Sacro Cuore, Rome) – Fig.7 Electrophoresis on agarose gel of RFLP RT-PCR for FLT3 D835 mutation. M = molecular weight; wild = wild type sample; Mt = mutant sample (image from Yamamoto et al, 2001).


NPM1 mutations

Nucleophosmin gene (NPM1) codifies for a protein, which works as shuttle between nucleolus, nucleus and cytoplasm, interacts with several proteins as molecular chaperone, controls ribosome formation and export, stabilizes p14Arf protein in the nucleolus and regulates centrosome duplication.

NPM1 mutations occur in 30% of AML (50-60% of AML with normal karyotype), are mutually exclusive with other recurrent genetic abnormalities and leads to cytoplasmic delocalization of nucleophosmin, detectable by immunohistochemistry (Falini B et al, 2005)PubMed. The WHO Classification (2008) identifies AML with NPM1 mutations as provisional category among AML with recurrent genetic abnormalities, which is associated with distinct genetic, pathologic, immunphenotypic and prognostic features.

In particular, AMLs with NPM1 mutations associate with CD34 negativity, normal karyotype in 85% cases, morphological variability with multilineage involvement, higher frequencies among de novo AML and in adults and relative favorable prognosis in absence of FLT3 ITD mutations. Approximately 50 molecular variants of NPM1 mutations have been described. Type A mutation occurs in 75% cases and consists in TCTG tetranucleotide insertion at 956 position of the terminal exon 12, with loss of tryptophan residues essential for the correct protein nucleolar localization and consequent formation of a C-terminal signal which drives the nucleophosmin cytoplasmic dislocation. Type B and C mutations consist in the insertion of a tetranucleotide CATG or CCTG in position 959 and occur in 15-20% of cases. NPM1 mutations are stable along the disease course and can be used as marker for minimal molecular disease monitoring.

Several assays are currently used for NPM1 mutation identification, utilizing as substrate either DNA or RNA (Table 5). They differ in diagnostic sensitivity, intended as the ability to identify most number of different mutations, and in analytic sensitivity, intended as blast percentage needed to get a positive result. Allele-specific oligonucleotide PCR (ASO-PCR), PCR with high resolution melting analysis, quantitative PCR and RT-PCR associate with high analytic sensitivity and can be used to study the minimal molecular disease (Wertheim G et al, 2008)PubMed.



Table 5C: Reported assays for NPM1 mutation screening (da Wertheim et al, 2008)


Immunohistochemical identification of nucleophosmin cytoplasm dislocation on BM trephine biopsy samples is highly predictive for presence of NPM1 mutation and represent a reliable, easy and cheap diagnostic surrogate of molecular studies, but cannot be used for minimal molecular disease monitoring.


CEBPA mutations

CEBPA gene codifies for the transcription factor CCAAT/enhancer binding protein alpha, which plays a pivotal role in granulocytic differentiation. CEBPA gene mutations occur in 5-14% AML patients and are commonly associated with a favorable outcome. Mutations can affect the N-terminal portion with abolishment of synthesis of 42 kDa working isoform (p42) in favor of synthesis of the dominant negative 30 kDa isoform (p30). Alternatively, mutations can affect the C-terminal portion with inhibition of DNA-binding activity and of protein homo- and heterodimerization. Rarely, mutations can occur at germinal level and cause familial AML cases. WHO classification of hematopoietic tissue neoplasm identifies AML with CEBPA mutation as distinct provisional nosographic category. In the common AML diagnostic work-up, CEBPA mutation search is relevant for prognostic stratification of patients with intermediate cytogenetic risk and normal karyotype, with a 5-year overall survival of 65% for double mutant patients (CEBPAdm), 56% for single mutant patients (CEBPAsm), and 39% of wild type patient (CEBPAwt). Prognosis in CEBPAsm is influenced by FLT3 and NPM1 genotype, while CEBPAdm genotype is rarely associated to the presence of other gene mutations and is considered a distinct nosographic category with a specific gene expression profile (Wouters et al, 2009; Taskesen Eet al, 2011 PubMed ). CEBPA mutation search is performed by direct sequencing of codifying region, consisting in one exon, through 3 or 4 distinct PCRs. Screening methods as the denaturing high performance liquid chromatography (dHPLC) and the fragment length analysis by PCR with capillary electrophoresis (PCR-CE), can identify amplicons carrying mutations which should undergo sequencing, allowing the reduction of number and costs of sequencing (Wouters et al, 2009; Fuster O et al, 2012PubMed ).


WT1 expression

Minimal residual disease (MRD) monitoring by RQ-PCR after induction and consolidation chemotherapy and after stem cell transplantation significantly affects the risk of relapse in AML patients. Nevertheless, most AML patients miss a molecular marker, such as a fusion gene, for MRD monitoring. Wilms tumor gene (WT1) is overexpressed in around 90% AML patients and its expression level are 2 log higher than the highest levels of normal control in 46% and 13% of AML PB and BM samples, respectively. For these patients, quantitative WT1 expression by RQ-PCR on PB or BM samples represents a useful marker of MRD. The European LeukemiaNet (ENL) MRD Workpackage evaluated 10 different RQ-PCR assays for WT1 and selected a standardized test with higher reliability. A WT1 transcript reduction of 2 log or higher after induction and consolidation therapy, compared to pretreatment levels, is associated to a lower risk of relapse. In particular, the entity of WT1 reduction after induction is an independent predictive factor for relapse (Cilloni D et al, 2009)PubMed .


JAK2 mutations

JAK2 gene localizes on chromosome 9p24 and codifies for a cytoplasmic tyrosine kinase that plays a crucial role in signal transduction of multiple growth factors.

JAK2 V617F mutation at level of exon 14 is common in Ph- Myeloproliferative Neoplasm and in particular occurs in 96% of patients with Polycythemia Vera, in 55% of patients with Essential Thrombocythemia and in 65% cases of Primary Myelofibrosis (Baxter et al, 2005). Less commonly JAK2 V617F mutation occurs also in other neoplasms of the hematopoietic tissue such as, Myelodysplatic Syndromes and Acute Myeloid Leukemias.

This mutation affects the pseudokinase non-catalytic domain of JAK2 and disrupts its regulatory activity. It can occur in homozygosis following mitotic recombination in patients with Polycythemia Vera and Primary Myelofibrosis. A higher allelic burden is associated the heavier constitutional symptoms and evolution in Myelofibrosis for patients with Polycythemia Vera and can be used to evaluate treatment response, but do not affect thrombotic risk, survival and evolution to AML.

JAK2 mutations at exon 12 occur in 2% patients with Polycythemia Vera and exclusively in those without the V617F mutation. They are commonly associated to isolated erythrocytosis, younger age and low serum erythropoietin levels.

Due to its high prevalence in Myeloproliferative Neoplasms, JAK2 V617F mutation is a useful marker of clonality. Screening for JAK2 V617F mutation is recommended in diagnostic work-up of erythrocytosis, thrombocytosis, splanchnic venous thrombosis and BCR-ABL negative granulocytosis not otherwise justified.

JAK2 V617F mutation is visualized by an allele specific oligonucleotide PCR (ASO-PCR) which uses a common reverse primer and two different forward primers: the former is specific for the mutant allele with a mismatch in the third nucleotide at 3’ end to increase the specificity (amplicon of 203 bp), the latter amplifies both the mutant and the wild type allele (amplicon 364 bp) and works as internal control (Fig. 8) (Baxter EJ et al, 2005)PubMed.

JAK2 exon 12 mutations are studied by PCR amplification followed by direct sequencing. They should not be routinely searched but only in patients with Polycythemia Vera or Idiopathic Erythrocytosis negative per V617F mutation.


c-MPL mutations

Mpl (myeloproliferative leukemia virus oncogene) gene maps on chromosome 1p34 and codifies for the thrombopoietin receptor. The W515L/K Mpl mutation at level of exon 10 was described in 3% patients with Essential Throbocythemia and 10% patients with Primary Myelofibrosis, while the S505N Mpl mutation was described in cases of Familial Thromocytosis. Both mutations results in constitutive activation of thrombopoietin receptor leading to megakaryocyte proliferation. The most commonly used method for searching Mpl exon 10 mutation is direct sequencing with Sanger method. The low incidence of Mpl mutations do not justify the routine screening in the work-up of Myeloproloferative Neoplasms, but cases with uncertain diagnosis. (Tefferi A et al, 2011)PubMed .



FIP1L1-PDGFRA fusion gene arises from cryptic deletion at level of chromosome 4p12 and occurs in Chronic Eosinophil Leukemia, and less commonly in rare cases of Acute Myeloid Leukemia and T cell Lymphoblastic Leukemia/Lymphoma with eosinophilia. The WHO classification identifies Myeloid/Lymphoid Neoplasms with Eosinophilia associated to FIP1L1-PDGFRA as new nosograohic category. Several alternative fusion partners of PDGFRA have been described, such as genes KIF5B, CDK5RAP2, STRN, ETV6 and BCR. Both FIP1L1-PDGFRA and the other molecular variants are inhibited by imatinib at lower concentration than BCR-ABL. The presence of FIP1L1-PDGFRA fusion gene is evidenced by nested RT-PCR (Fig. 9) (Cools J et al, 2003)PubMed.


Mutazione c-Kit D816V

The proto-oncogene c-Kit codifies for a type III trans-membrane tyrosine kinase receptor, whose ligand is the Stem Cell Factor. After binding with its ligand, c-Kit dimerizes and undergoes autophosphorylation with following activation of intrinsic tyrosine kinase and phosphorylation of downstream proteins, leading to cellular proliferation, maturation and survival. c-Kit mutations have been described in several neoplasms.

The activating D816V c-Kit mutation occur in 80% cases of Systemic Mastocytosis and represents a potential therapeutic target. Moreover the identification of c-Kit D816V mutation is clinically relevant because is one of minor criteria for the diagnosis of Systemic Mastocytosis. Patients carrying this mutation do not respond to imatinib treatment, but can be still sensitive to dasatinib. c-Kit mutations, including both D816V mutation and exon 8 mutations, have been described also in 2-5% AML and up to 12-48% of Core-Binding Factor (CBF) AML where they assume a poor prognostic value.

Several assays have been tested to search the c-Kit D816V mutation. Direct sequencing and denaturing high-performance liquid chromatography (HPLC) are not routinely used due to the low sensitivity. Other methods include the PNA (peptide nucleic acid) directed PCR clamping, PCR followed by enzymatic restriction and the allele-specific oligonucleotide PCR (ASO-PCR). This last assay uses two couples of oligonucleotides for two distinct PCRs: the former reaction amplifies a portion of c-Kit exon 17 independently of the mutation and works as internal control; the latter uses a mutant allele-specific forward primer which matches at its 3’ end only with the mutant sequence and allows the amplification of the mutant allele but not the wild type. ASO-PCR is very sensitive and specific for c-Kit D816V mutation screening and can identify the mutation in presence of less than 1% tumor cells (Fig. 10) (Schumacher JA et al, 2008)PubMed.


Zini_piattaforme_di_diagnosi_in_ematologia_Figura_9 Zini_piattaforme_di-diagnosi_in ematologia_Figura_8Zini_piattaforme_di_diagnosi_in_ematologia_Figura_10

Fig.9C                                                          Fig.9C                                               Fig.10C

Fig.8 Electrophoresis on agarose gel of PCR products for JAK2 V617F mutation detection. Wild type samples present an unique 364 bp amplicon (lanes 6 and 8-12), while mutant samples show 2 amplicons of 364 bp and 203 bp, respectively (samples 1-5 and 7). PV= Polycythemia Vera; ET = Essential Thrombocytemia; IMF =Idiopathic Myelofibrosis (image from Baxter et al, 2005) – Fig.9: Electrophoresis on agarose gel of nested RT-PCR for FIP1L1-PDGFRA. Lane 1: 100 bp molecular weight; lanes 2 and 3: positive samples; lane 4: positive control; lane 5: negative control. (Courtesy of Dr F. Guidi, Institute of Hematology, Università Cattolica del Sacro Cuore, Rome) – Fig.10 ASO-PCR for c-Kit D816V mutation detection. Patient’s sample carrying this mutation amplifies a 184 bp product corresponding to the wild type allele and a 90 bp product corresponding to the mutant allele (Courtesy of Dr F. Guidi, Institute of Hematology, Università Cattolica del Sacro Cuore, Rome).


Immunoglobulin and T-cell receptor gene rearrangement analysis

In most patients with suspected lymphoproliferative diseases, histology and cytology combined with immunohistochemistry and immunophenotype in flow cytometry are sufficient to distinguish between lymphoproliferative neoplasm and reactive lymphoid proliferation. In a minority of cases, diagnosis needs to be supported by lymphocyte clonality assays, which are founded on the premise that all tumor cells arise from the same clonal cell.

During lymphocyte proliferation, the rearrangement of gene codifying for immunoglobulin (Ig) and T-cell receptor (TCR) produces unique DNA sequences. Almost all lymphoid neoplasms have clonally rearranged sequences of Ig or TCR, which can be utilizes as markers of clonality.

The loci for Ig and TCR genes contain multiple different genetic sequences named variable (V), diversity (D), and joining (J), which undergo processes of recombination by RAG1 and RAG2 enzymes during the early phases of lymphocyte differentiation. Genes for Ig heavy chain (IgH), TCR beta (TCRB) and TCR delta (TCRD) produce V-D-J rearrangements, while gene for Ig light chain kappa (IgK) and lambda (IgL), TCR alpha (TCRA) and TCR gamma (TCRG) give rise to V-J rearrangements. DNA sequences between rearranged genes are deleted. Possible recombinations between V-D-J genetic sequences are calculated around 2 x106 for Ig, 3 x106 for TCRαβ, and 5 x106 for TCR γδ. Random single nucleotide insertion or deletion at junction sites of VDJ sequences further increases the recombination repertoire to more than 1012. Moreover the somatic hypermutation process that occurs in B lymphocytes of germinal center after antigen presentation, further contributes to DNA sequence diversification through single nucleotide substitutions, deletions or insertions.

Molecular assays for B and T cell clonality are recommended in the following situations: in case of suspected B cell neoplasm if morphology and immunophenotype are not conclusive; in all cases of T cell proliferation; in case of lymphocyte proliferation in immunocompromised patients, in particular after transplantation; to establish clonality relation between two distinct lymphoid neoplasms in the same patients, to discriminate between relapse and secondary cancer; occasionally to complete lymphoma classification and stadiation.

Southern blot was used in the past to asses Ig and TCR gene clonality but in the last two decades has been replaced by Polymerase Chain Reaction (PCR), which requires fewer DNA amount and of inferior quality, allowing clonality studies also in small tissue samples and biologic fluids or in paraffin-embedded samples.

Limits of these assays are related to the percentage of normal lymphocytes in which the neoplastic population is diluted, with a sensitivity between 1 and 10% to identify clonal cells. Moreover, clonality is not synonymous of malignancy and cross-lineage clonal rearrangements have been described in several tumors. The most commonly used PCR assays for B and T cell clonality concern IgH and TCRG genes. Almost all T cell lymphomas (>98%) have clonally rearranged TCRG gene, although expressing TCRαβ.

The BIOMED-2 Concerted Action BMH4-CT98-3936 standardized PCR protocols and primer sets for clonality diagnostic assays (Fig. 11) (van Dongen JJ et al, 2003)PubMed. For IgH gene complete rearrangement, three different multiplex PCRs (FR1, FR2 and FR3) are used with three different primer sets that bind VH sequences, and a single consensus primer that binds a high homology region on JH gene. Three different multiplex PCR are needed to study TCRB rearrangements, two multiplex PCRs to study the incomplete IgH DH-JH rearrangement and the IgK and TCRG rearrangements, while only one multiplex PCR is sufficient to study IgL and TCRD rearrangements. PCR products are visualized by heteroduplex analysis or GeneScanning. The heteroduplex analysis is performed through high temperature denaturation of PCR products followed by rapid and casual low temperature denaturation. This process causes the formation of several heteroduplexes with different migration speed in case of polyclonal lymphocyte proliferation, while it leads to formation of homoduplexes with identical migration rate in case of clonal lymphocyte proliferation. The race of PCR products on 6% polyacrylamide gel shows a single band in case of homoduplex, while heteroduplexes produce a smear higher in size (Fig. 11b).

GeneScanning analysis of PCR products uses fluorochrome-labelled primers and automatic sequencer. Labelled single strand PCR products are separated on polymer for capillary sequencing and analyzed by automatized laser scanning. Polyclonal lymphocyte proliferations results in a Gaussian distribution of multiple spikes, while clonal proliferations give rise to a single spike corresponding to a unique PCR (Fig. 11c). Guidelines for using clonality assays have been recently codified by the International Consortium EuroClonality/BIOMED-2 which are summarized in the Figure 12  (Fig. 12) (Langerak et al, 2012).

Sequencing of PCR products arising from clonal rearrangements of Ig and TCR genes allows to design specific forward primers that bind patient’s unique V-N-D-N-J or V-N-J sequences. These allele-specific oligonucleotides, in combination with probes and reverse primers binding germline J sequences, allow to monitor the lymphoproliferative disease through an allele specific real time quantitative PCR  (ASO-RQPCR). The ASO-RQPCR has a diagnostic sensitivity of 10-4 – 10-5 and is performed on both BM and PB samples. Molecular disease monitoring by ASO-RQPCR after chemotherapy and after hematopoietic stem cell transplantation has been shown to predict relapse risk, progression-free survival (PFS) and overall survival (OS) of patients affected by several types of lymphoproliferative disease, including Acute Lymphoblastic Leukemia, Chronic Lymphatic Leukemia, Plasma cell Myeloma, Follicular Lymphoma and Mantle Cell Lymphoma (Brüggemann M et al, 2006 PubMed ; Dreger P et al, 2005PubMed ; Ferrero S et al, 2011PubMed ; Pott C et al, 2010PubMed ).

Besides diagnostic purposes, the study of the Ig heavy chain rearranged gene sequences significantly contributed to increase the knowledge about the origins of lymphoproliferative neoplasms. In particular, in specific subtypes of lymphoid neoplasms the non-random stereotyped selection of genes for the variable regions of Ig, which contribute to the Complementary Determining Regions (CDR) of Ig molecules, supported the hypothesis of an antigen-driven selection of the neoplastic clone.

Moreover, the analysis of somatic hypemutations at level of the rearranged variable regions of Ig allowed to define the origin of malignant clonal lymphocytes as pre-germinal center, germinal center or post-germinal center. Gene sequences for the rearranged variable regions of Ig heavy chain (IgVH) have been defined as mutated or unmutated according to the presence of somatic mutations more or less than 2% in comparison to the germ line sequences. In the context of Chronic Lymphatic Leukemia, the analysis of the mutational IgVH status is routinely performed and the presence of unmutated IgVH represents a negative prognostic factor. The IgVH mutational status analysis is performed on genomic DNA or more commonly on cDNA arising from RNA retrotranscription, though a multiplex PCR using a mix of forward primers specific for each leader sequence of VH1-VH6 families, together with a mix of reverse primers complementary to germ line JH sequences or to the constant region. PCR product are sequenced with automated sequencers and the nucleotide sequences aligned with the germ line sequences available on nucleotide databases (EMBL/GenBank o V-BASE sequence directory). Sequences with homology higher than 98% with germ line sequences are considered unmutated (Hamblin TJ et al, 1999)PubMed .



The t(11;14)(q13;q32) translocation is characteristic of Mantle Cell Lymphoma (MCL) and occurs sporadically in other B-cell lymphoproliferative neoplasms. The breakpoints are localized in a 360 kb region at 5’ of cyclin D1 gene (CCND1). In half of MCL cases, the breakpoint occurs at level of a 85 bp region named major translocation cluster region, BCL1-MTC. In most cases the breakpoint on IgH locus at 14q32 involves JH genes, juxtaposing the IGH-Eµ enhancer close to the sequence on chromosome 11q13, with following transcriptional activation of cyclin D1 gene. Together with CDK4, cyclin D1 phosphorylates and inactivates pRB and allows cell cycle progression through G1 phase. Cyclin D1 is not expressed by normal B lymphocyte and by malignant lymphomas other than MCL. Since the presence of translocation t(11;14)(q13;q32) correlate with cyclin D1 expression, both the expression and the translocation are additional elements for differential diagnosis among B cell lymphomas. The gold standard for t(11;14) identification is the FISH on interphase, able to identify the translocation in almost all MCL cases. PCR-based techniques are able to identify only 40% cases with high percentage of false negative, but can be used to monitor MRD (Fig. 13)(van Dongen JJ et al, 2003)PubMed.


Zini_piattaforme_di_diagnosi_in_ematologia_Figura_11 Zini_piattaforme_di_diagnosi_in_ematologia_Figura_12 Zini_piattaforme_di_diagnosi_in_ematologia_Figura_13

Fig.11C                                                         Fig.12C                                             Fig.13C


Fig.11 Schematic representation of heteroduplex and GeneScanning analysis of PCR products resulting from Ig and TCR gene rearrangements (image from Van Dongen et al, 2003) – Fig.12 EuroClonality/BIOMED-2 multiplex PCR protocol for clonality PCR-based assays in suspected lymphoproliferative neoplasms with uncertain diagnosis or uncommon for histology, immunophenotype or clinical presentation (Image from Langerak et al, 2012) – Fig.13 Electrophoresis on agarose gel of PCR for t(11;14)(q13;q32) detection). Lane 1-5: negative samples; lane 6: positive sample; lane 7: positive control; lane 8: negative control (Courtesy of Dr F. Guidi, Institute of Hematology, Università Cattolica del Sacro Cuore, Rome).



The translocation t(14;18)(q32;q21) occur in about 90% of Follicular Lymphomas and in 20% of Diffuse Large B Cell Lymphomas. The detection of this translocation is important both for the diagnosis and MRD monitoring. Followiing the translocation the antiapoptotic gene BCL2 on chromosome 18q21 is juxtaposed close to the enhancer of IgH locus on chromosome 14q32.3, resulting in deregulation of its expression.

The breakpoint on IgH locus occurs during the VDJ recombination process and one out of the six germ line JH regions become strictly juxtaposed to the BCL2 gene. The breakpoints on chromosome 18q21 are variable. Most breakpoints occur in a 159bp region named Major Breakpoint Region (MBR), located at 3’ non-translated region of exon 3. Another breakpoint region is located 4 kb downstream of MBR, is named 3’MBR and spans 3.8 kb. The minor cluster region (mcr) is located 20 kb at 3’ of MBR and spans a region of 500 bp. Other breakpoints have been described at 5’ of BCL2 gene but are rare in frequency and are not routinely amplified by PCR

PCR-based techniques are able to identify only 60% of translocation t(14;18), with high rate of false negative (van Dongen JJ et al, 2003)PubMed. These assay are of easy applicability and allow to monitor MRD (Fig. 14 e Fig. 15).


BRAF V600E mutation

Whole exome sequencing of leukemic cells and corresponding normal cells in an index patients with hairy cell leukemia (HCL) led to the identification of a heterozygous point mutation at level of exon 15 of BRAF oncogene with substitution of glutamic acid in the place of valin in position 600 (V600E). This mutation was previously described in melanomas and papillary thyroid cancers and has been confirmed in 100% cases of HCL, while is absent in other B-cell neoplasms including splenic marginal zone lymphoma, splenic red pulp lymphomas and variant HCL (Tiacci E et al, 2011)PubMed .

The V600E mutation leads to the constitutive BRAF activation with following phosphorylation of downstream targets MEK and ERK, which contribute to increased cellular proliferation and neoplastic transformation.

Sequencing technique has low sensitivity to identify BRAF V600E mutation, requiring at least 30% of circulating leukemic cells in PB. For routinely diagnostic work-up, an easy and cheap ASO-PCR has been developed with 100% diagnostic specificity and 0.1% sensitivity for mutant allele detection (Tiacci E et al, 2012)PubMed . This assay consists of two distinct PCRs that have a common reverse primer but differ for the forward primer, the former specific for the wild-type allele and the latter for the mutant allele. PCR product are visualized by electrophoresis on agarose gel.


MYD88 L265P mutation

Whole genome sequencing of lymphoplasmocytic cell DNA from 30 patients affected with Waldenstrom’s Macroglobulinemia led to the identification of a somatic mutation (T→C) in position 38182641 on chromosome 3p22.2 corresponding to the aminoacid substitution L265P of MYD88 gene.

This mutation, confirmed by Sanger sequencing, was found both in BM and PB malignant lymphoplasmocytic cells, but not in the same patients’ normal tissues. It occurs in 91% patients with Waldenstrom’s Macroglobulinemia and non-IgM secreting lymphoplasmocytic lymphoma, while it’s rare or absent in other subtypes of lymphoproliferative neoplasms, such as Plasma cell Myeloma, Marginal Zone Lymphoma, IgM monoclonal gammopathy of unknown significance (Treon SP, et al 2012)PubMed .

This mutation was described also in 29% of activated B cell-type DLBCL, but was absent in other DLBCL subtypes and in Burkitt’s Lymphoma. The L265P MYD88 mutation plays an important pathogenetic role because promote cell survival assembling a protein complex containing IRAK1 e IRAK4, with following activation of IRAK4 kinase, IRAK1 phosphorylation, signaling through NF-κB, STAT3 actvation by JAK and IL-6, IL-10 and interferon-β secretion (Ngo VN et al 2011)PubMed .

Beside Sanger sequencing approach, several methods are available for an easy, quick and cheap detection of MYD88 mutation, including PCR-RFLP, high-resolution melting curve analysis, mismatch PCR-RFLP (Gachard N et al 2013 PubMed ; Wang et al 2012; Guidi and Hohaus, unpublished methods, Fig. 16). The finding of MYD88 L265P mutation can represent a helpful diagnostic tool for differential diagnosis between borderline lymphoproliferative disorders such as Waldenstrom’s Macroglobulinemia/lymphoplasmocytic lymphomas, marginal zone lymphomas and lymphocytic lymphoma, as well as could have future therapeutic implications for target therapy.


Zini_piattaforme_di_diagnosi_in_ematologia_Figura_14 Zini_piattaforme_di_diagnosi_in_ematologia_Figura_15 Zini_piattaforme_di_diagnosi_in_ematologia_Figura_16

Fig.14C                                                         Fig.15C                                             Fig.16C


Fig.14 Electrophoresis on agarose gel of PCR for t(14;18)(q32;q21) MBR rearrangement. Lane 4: positive sample; lane 1-3, 5 and 6: negative samples; lane 7: positive control; lane 8: negative control; lane 9: 100 bp molecular weight (Courtesy of Dr F. Guidi, Institute of Hematology, Università Cattolica del Sacro Cuore, Rome) – Fig.15 Electrophoresis on agarose gel of PCR for t(14;18)(q32;q21) mcr rearrangement. Lane 1: positive sample; lane 2-6: negative samples; lane 7: positive control; lane 8: negative control; lane 9: 100 bp molecular weight (Courtesy of Dr F. Guidi, Institute of Hematology, Università Cattolica del Sacro Cuore, Rome) – Fig.16 Mismatch PCR-RFLP for MYD88 L265P mutation detection. Lane 1: 100 bp molecular weight; lanes 2 and 5: mutant samples; lanes 3 and 4: wild type samples; lane 6: negative control (Courtesy of Dr F. Guidi and Dr S. Hohaus, Institute of Hematology, Università Cattolica del Sacro Cuore, Rome).


Gene expression profiling

Tumors of hematopoietic and lymphoid tissue arise from multiple genetic and epigenetic lesions acquired at somatic level by the hematopoietic stem/progenitor cells, finally resulting in deregulated gene expression. The identification of specific genetic and molecular abnormalities contributes to the actual classification of these neoplasms and assumes a crucial role in planning the treatment and patients’ prognostic stratification.

At the beginning of 2000s, the coming of new technologies for the study of genome allowed a better characterization of molecular abnormalities which account for myeloid and lymphoid tumor clinical and biological heterogeneity.

The gene expression profiling (GEP), founded on the microarray technology, identified disease-specific signatures by simultaneously evaluating the expression of thousands of genes. In particular, the GEP allowed the characterization of gene expression patterns in myeloid and lymphoid neoplasms subtypes associated to distinct genetic and/or molecular abnormalities, the identification of new biological entities and the prediction of response to standard therapy. Moreover, GEP has given new insights in pathogenesis of hematological malignancies and identified molecular target for new therapeutic approaches (Theilgaard-Mönch K et al, 2011)PubMed. Although several microarray technologies and platforms have been used, the GEP methods can be standardized for diagnostic purposes, because distinct gene expression patterns have been highly reproducible within the same platforms and among different platforms.

The collaborative study program Microarray Innovations in Leukemia (MILE), associated to the international group European LeukemiaNet, which includes 11 laboratories of three different continents, evaluated the clinical accuracy of gene expression profiles of 16 subtypes of acute and chronic leukemias, myelodysplastic syndromes and a control group including normal and non-malignant BMs, using current diagnostic routine algorithms. In the first cohort of 2096 patients, GEP produced a diagnostic accuracy of 92.2% for 18 distinct disease subtypes with a median specificity of 99.7%. In the second cohort of 1152 patients taken in prospective manner, GEP-based classification reached a median sensitivity of 99.7% and a median specificity of 99.8% for 14 acute leukemia subtypes (Haferlach T et al, 2010)PubMed.

GEP is able to predict the presence of recurrent genetic abnormalities in patients affected by Acute Myeloid Leukemia, such as t(8;21), inv16, t(15;17), MLL gene abnormalities/t(11q23), and CEBPA and NPM1 mutations. Moreover, through GEP studies a panel of 133 genes able to define patients’ prognosis has been identified (Bullinger L et al, 2004)PubMed.

GEP studies on Myelodysplastic Syndromes has been performed both on immunoselected CD34+ of CD133+ cells, and on unfractionated mononuclear BM cells, with heterogeneous results compared to AML. However GEP studies demonstrated deregulation of genetic pathways linked to interferon signaling, thrombopoetin and Wnt pathway. Low-risk MDS presented deregulation of genes involved in immune surveillance, apoptosis and chemokine signaling, while high-risk MDS were characterized by abnormalities of DNA damage response and cell cycle checkpoint. Distinct gene expression profiles have been described in patients with recurrent genetic abnormalities, such as 5q-, trisomy 8 and 7-/del(7q). In the MILE study, diagnostic GEP accuracy was only 50% on unfractionated BM mononuclear cells from MDS patients but allowed the identification of genetic signatures with prognostic value associated to higher risk of AML transformation (Theilgaard-Mönch K et al, 2011)PubMed.

GEP use is so far confined to clinical trials and is not routinely performed in the diagnostic algorithm of neoplasms of hematopoietic tissue. Although it has huge potentiality to identify multiple genetic/molecular abnormalities for the classification and prognostic stratification of leukemias, this technology is continually evolving and needs a significant technical and analytical support. Moreover, further studies are needed to give the right weight to the huge amount of data resulting from GEP analysis.


FISH (Fluorescence In Situ Hybridization)

FISH analysis use DNA probes with fluorochromes complementary to specific genomic sequences. When we identify the gene or the chromosomal segment to test, we proceed to DNA denaturation in presence of specific fluorescent probe. After this hybridization, we analyze the nucleus in fluorescence microscopy. Several tissue samples are analyzed with this method, mainly from peripheral blood or bone marrow, but also liquor, ascitic fluid and formalin-fixed paraffin-embedded tissue. We can detect both loss and gain of chromosomal DNA in the cell; we can detect also specific recurrent translocation. To identify trisomy/monosomy we use specific probes to chromosomal centromere (centromeric probe, e.g. cr8 or cr12), whereas to detect deletion or acquisition in single gene or chromosomal portion we use gene- or segment-specific probes (e.g. del13q14, del17p13 or del5q).

In case of balanced translocations (e.g. BCR/ABL in chronic myeloid leukemia or PML/RARα in promyelocytic leukemia) the two genes involved are marked with two different fluorescent probes (red-green); the cells with translocations have two yellow fusion signals (“dual-color-dual-fusion” probe). The “dual-color-break-apart” probe shows the gene translocation if we don’t know the partner-gene: the C-terminal and the N-terminal segment of the gene have two different fluorochrome (red or green) and they are singularly visible after gene translocation (e.g. RARα gene in promyelocytic leukemia).

We can use FISH analysis not only in metaphase nucleus (like classical cytogenetic procedures), but also in interphase nucleus. This peculiarity is important in hematological neoplasms without high mitotic index (chronic lymphocytic leukemia, lymphoma and multiple myeloma). FISH analysis is faster than classical cytogenetic, this feature is important when a prompt diagnosis change the prognosis (e.g. promyelocytic leukemia and treatment with retinoic acid.

This technic can show both loss/gain of genetical material and translocation of a specific gene, but highlighted point mutations aren’t highlight: in this case we use molecular biology. FISH sensitivity is higher than sensitivity of conventional cytogenetic (we analyse 200-500 nuclei), but is inferior to sensitivity of molecular biology (sens 1:10,000). Therefore we use this approach at diagnosis or during relapse, with an high number of neoplastic cells containing specific aberration; for minimal residual disease determination molecular biology (RT-PCR) is preferable.

The whole genomic material can be potentially analyzed by FISH, but in clinical routine we use the specific loci more probably involved in the suspected disease.



Chronic myeloid leukemia (CML)

In the diagnosis of CML the element key is the identification of balanced translocation t(9;22) with generation of fusion gene BCR/ABL. “LSI bcr/abl dual fusion DNA probe” bind chromosomal segment 22q11.2 (breakpoint cluster region Green) and segment 9q34 (abl oncogene Red). In the normal nucleus in interphase we identify two green signals and two red; in pathological cells we see only one red and green signal and two fusion signals red-green (derivative chromosome 9;22). (Fig. 1)


Philadelphia-negative chronic myeloproliferative neoplasms

In this neoplasms, once excluded t(9;22), we can identify several cytogenetic aberrations; they are present in approximately 30% of myelofibrosis an polycythemia, more rare in essential thrombocythemia (69, 70); the most frequent are trisomy of cr8 or cr9 and 13q and 20q deletion. However, at diagnosis, we use classical cytogenetic and FISH analysis to confirm the results.


Myelodisplastic syndrome (MDS)

With classical cytogenetic we identify chromosomal abnormalities in approximately 50% de novo-MDS and in 80% therapy-related MDS (2). In the classical cytogenetic the whole genome is investigated; in the FISH analysis, on the contrary, we investigate the chromosomal segment bind to the probe. Therefore, we search only more frequent aberrations: trisomy of the cr8, monosomy cr5/5q deletion, monosomy cr7/7q deletion, 20q deletion, -Y.

FISH is important in case of absence of evaluable metaphase in classical cytogenetic; its role is debatable in normal kariotypes (71,72,73) (in literature, 15% of MDS with normal karyotype in classical cytogenetic have some alterations in FISH analysis). (73,74)

To identify cr5 and cr7 monosomy or 5q- and 7q-deletion we use specific probes complementary to cr5/7 centromeres (5p15.2 and 7p11.1-q11.1, green) and complementary to 5q and 7q segment (5q31 and 7q31, red). Monosomic cells have only one red and green signal; the cells with 5q or 7q deletion have two green signals, but only one red. (Fig. 2)

In the case of cr8 trisomy we see three fluorescent signals when we use a specific centromeric probe. (Fig 3)

The fluorescence signal specific to Y centromere is lost in the cells with -Y.

The deletion of 20q is detected by specific probe who bind 20q12; the pathological cells have only one red fluorescent signal.


Zini_piattaforme_di-diagnosi_in ematologia_Figura_1D Zini_piattaforme_di-diagnosi_in ematologia_Figura_2DZini_piattaforme_di-diagnosi_in ematologia_Figura_3D

Fig.1D                                                          Fig.2D                                              Fig.3D


Chronic lymphocytic leukemia (CLL)

Approximately 80% of patients with CLL has genomic abnormalities. In some cases we have a worst prognosis (e.g. del17p or del 11q) and some therapeutic implications (a worst response to alkylating agents and to purine analogs in cases with del17p); to date, this analysis is recommended before treating this patients.

The most frequent alteration is the 13q deletion (almost 50% of cases); the specific probe bind 13q14 segment; the deletion is detected by identification of one only signal (not two) in the cell (Fig. 4)

Similarly, one only signal for 11q22 segment detect 11q deletion (with ATM loss). When we use a specific probe to chromosome 12 centromere, we identify the cells with cr12 trisomy.

In 17p deletion, we have a deletion of p53 gene (oncosuppressor gene). The specific probe brand in red the 17p13 segment; in this case, we use also a green probe to cr17 centromere.


Non-Hodgkin lymphoma (NHL)

Although molecular biology is more sensible than FISH in this case, we can detect several recurrent abnormalities. FISH analysis can detect t(14;18) in follicular lymphoma, t(11;14) in mantle cell lymphoma (Fig. 5) or t(8;14) in the Burkitt lymphoma (Fig.6); in the Burkitt lymphoma we can detect also t(8;2) o t(8;22) (in all cases, MYC gene is involved). In all cases we use dual color-dual fusion probes ( like t(9;22)). In t(8;14) the implicated segments are 14q32 and 8q24; in t(11;14) are 14q32.3 and 11q13 and in t(14;18) are 14q32 and 18q21.


Zini_piattaforme_di-diagnosi_in ematologia_Figura_4D  Zini_piattaforme_di-diagnosi_in ematologia_Figura_5D Zini_piattaforme_di-diagnosi_in ematologia_Figura_6D

Fig.4D                                                         Fig.5D                                             Fig.6D


Multiple Myeloma (MM)

Plasma cells have a low mitotic index, like lymphocytes in CLL; for this reason, FISH analysis is the most important technique to detect genomic abnormalities. To increase the sensibility, we must perform this analysis with purified cells or combining FISH analysis with fluorescent superficial light chain of the plasma cells.

Similarly to CLL, we can detect del13q or del17p; the first one is frequent, but currently without a significant prognostic role, the second associated to worst prognosis.

Main translocations in MM involve the heavy chain locus in 14 chromosome; partners are 11q23 segment in t(11;14), 4p16 in t(4;14) and 16q23 in t(14;16).


Acute lymphoblastic leukemia (ALL)

FISH can detect t(9;22); although breakpoint in BCR gene is different respect to CML, the optical appearance is the same to BCR/ABL translocation in CML.

Especially in pediatric ALL, MLL gene can be involved in balanced translocation; but more than 50 translocation partners are identified. To date, we use a break-apart probe, where N-terminal and C-terminal segment bind a different fluorochrome (red and green); the normal gene has only one yellow signal, but in the translocation case, red and green signals are evident.


Acute myeloid leukemia (AML)

Conventional cytogenetic is the gold standard in AML: we have many genetic abnormalities: FISH analysis can detect recurrent and specific abnormalities (“acute myeloid leukemia with recurrent genetic abnormalities” in WHO classification). Dual color-dual fusion probes detect t(8;21) or t(15;17); break-apart probes detect inv16 or t(16;16), MLL or RARα translocation.


Allogenic stem cell transplantation

After sex-mismatched allogenic stem cell transplantation, engrafment and chimerism are monitored with FISH analysis of sex chromosome X and Y: the two centromeres have two different fluorochromes (red or green).


Bone marrow trephine biopsy

The BM trephine biopsy (TB) is an uncomfortable procedure which should be performed on specific clinical indications: a)patients with hypocellular marrow or with fibrosis; b) patients with inadequate or insufficient aspirate findings (punctio sicca); c) patients showing a focal pattern of infiltration such for granulomatous pathologies or lymphomas; d) patients with pancytopenia and leuco-erithroblastosis at the peripheral blood smear; e) patients with Myeloprolipherative Disease (MDs) (Ph -); f) patients with the suspect of non- hemopoietic malignancy infiltration; g) patient with multiple myeloma; h) patient with cytopenias of unclear cause. In particular, the TB should be considered in the LPDs to provide the initial staging of non Hodgkin lymphomas (NHL) and Hodgkin Lymphomas as well as the follow-up assessment of response to therapy in patients with stage IV disease. In patients with HL associated to cytopenias and B- symptoms or immune-deficiencies the BM involvements is substantially higher as well as the case of classic subtype. In multiple myeloma this procedure has an essential role in the diagnosis of patients with hypocellular marrow or small number of plasma cells at the aspirate due to a focal distribution. The BM examination plays an important role in the distinction of Polycythaemia Vera (PV), Essential Trombocythaemia (ET) and Primary myelofibrosis (PMF) among myeloproliferative neoplasms (Philadelphia negative). In particular, panmyelosis with normally folded or deeply lobulated megakaryocytes frequently distributed in loose clusters identify the PV and the pre-PV, while the increase number of enlarged mature megakaryocytes generally defines the ET. The pre-fibrotic phase of PMF appears with hypercellular BM and increased number of enlarged and small megakaryocytes with atypical nuclei (Fig.1). With the purpose to better identify megakaryocytes the use of megacaryocytic markers such as CD42 and CD61(Fig.2) are recommended. Diffuse and dense increase in reticulin with foci of immature cells (CD34 pos) are features of advanced PMF (MF-2 and MF-3)


Basic Panel: first level study

The screening in histopathology includes a basic panel of antibodies to identify the presence of pathology and the interested lineage. Set of antibodies such as CD3, CD19, CD79a and CD20 for the distinction of T- and B- lineage should be considered in LPDs. The transcript factor MUM1/IRF4 also add further information to immune- cytochemical diagnosis (Fig. 3) (Wilkins BS, 2009)PubMed . MUM1/IRF4 is a member of the IRF family of transcriptional factors and it plays a crucial role in the cell proliferation, differentiation and survival. MUM1 is mainly present in plasma cells and germinal center B cells located in the light zone. It appears to be strongly expressed in multiple myeloma, lymphoplasmacytic lymphoma , classical Hodgkin lymphoma and nearly one- half of diffuse large B-cell lymphomas, immunoblastic lymphoma but it seems to be absent in follicular and mantle cell lymphomas (Fig.3)(Falini B, 2000). Recent data explained as MUM1 can dichotomize Follicular lymphomas in low grade (MUM1 neg with positive CD10 and bcl-6 ) from high –grade FL (MUM1 pos with weaker expression of both CD10 and bcl-6 ) (Naresh KN, 2007). To this panel , CD30 antibody must be added the case of a suspect HL (Fig.4). In the screening of the MPDs, MDS and acute leukemias the CD34 antigen should always be added to allow a clearer distinction between three entities. In particular, in cases with insufficient specimen collected (punctio sicca . hypocellular marrow) CD34 antigen in histological specimens show an important usefulness for the diagnosis (Fig.5). The Myeloperoxidase (MPO) confirms the affiliation of the blasts to the granulocytic lineage . The presence small megakaryocytes makes difficult their identification to hematoxylin & eosina in both MPDs and MDS. Thus, markers as CD42 or CD61 provide a remarkable help to detect megakaryocytes (Wilkins BS, 2011) Pubmed . Macrophages infiltratrations can resemble to granulomatous disease and they are frequently source of confusion. The CD68 should be used for confirming the presence of macrophages although masts cells also express the same marker and CD117 antigen or the tryptase shoud be added to exclude these cells. Finally, when metastasys of solid tumor are suspected markers as CD45, CAM5.2, EMA, S100 and Melan A should be used.


Diagnosis: second level study

Once established the lineage of the pathological population more antigens should be studied with the purpose to reach the definitive diagnosis. Within B- LNH markers as CD5, CD23, CD10 may be added to the basic panel for completing the study. Bcl-6 is a nuclear transcription factor which suppresses p53 in B lymphocytes of the germinal centre (GC) and protects B cells from the apoptosis induced by DNA damages. The BCL6 gene also undergoes somatic mutation in the GC thus serving as a marker of cells that have been through the GC. This molecular marker is expressed by both centroblasts and centrocytes but it is switched off in memory B cells and plasma cells. It is over-expressed in most lymphomas showing cells that have been exposed to the GC such as diffuse large B-neoplasms (DLBCL), follicular and Burkitt lymphoma. On the other hand, the cyclin D1 plays an important role in the diagnosis of mantle cell lymphomas because the translocation t (11;14) is present in almost all cases and this cytogenetic abnormality results in deregulated overexpression of cyclin D1 (Fig. 7). The Ki-67 protein is a cellular marker of cell cycle and it is present during all active phases of the cell cycle (G1, G2, S and mitosis) but it is absent in the phase G0. These features makes Ki-67 an excellent marker to determine the growth fraction of a given cell population. The Bcl-2 is the founding member of Bcl-2 family of regulator proteins that regulate cell death. Indeed, tumours of GC B-cells (follicular lymphoma, sub-group of DLBCLs that originates from GC and 20% Burkitt ‘ s lymphoma) originate when GC cells fails to undergo apoptosis, in most cases due to a chromosomal rearrangement as t(14;18) that prevents the switch off of Bcl-2 protein expression. If Hairy Cell Leukemia is suspected CD25 and CD103 MoAbs must be added. In HLs the anti-CD30 and CD15 must always be added for confirming the presence of Reed Sternberg cells as well as the protein EBV-LMP1 should be investigated because represents one of the Ebstein Barr virus products which is essential for the neoplastic transformation . When Burkitt or lymphoblastic lymphomas are suspected the diagnosis of a B lymphoblastic leukemia should be excluded with the markers TdT and CD34. The protein PAX-5 also plays an important role in the proliferation and differentiation of B cells showing its usefulness to diagnose B-ALL with CD22 and cytoplasmic CD79a (Fig.8). Within T-lineage specific LDs followings antigens may be added to the basic panel: CD2,CD4, CD5, CD7, CD8 and CD25. In fact, lymphocytes are mature in the Sezary syndrome and they are generally positive to CD4 while they are missing of the CD7 (Fig.9). The CD30 antigen may be considered in T-lineage LPDs with the purpose to identify the anaplastic large cell lymphoma while the marker CD 10 is useful to identify the angioimmunoblastic T-cell lymphoma. It is important to underline that the T hepatosplenic T-cell lymphoma lacks in CD4 and CD8 antigens while it displays CD3 antigen and the γδ-receptor . The study of NK LPDs needs of the antigens CD56, CD57 associated to the presence of granzyme B. In multiple myeloma the basic panel should be extended with the CD138 and the CD56 which usually distinguish pathological plasma cells from the normal ones (Bayer-Gardner IB,2001). To detect the heavy chain specific antibodies can be added for the IgA, IgG, IgD (IgM) specificity (Fig.10) (Wilkins BS, 2009)PubMed.



Bone marrow trephine biopsy can be used to evaluate the response to the therapy. Different panels should be used on the basis of the pathology investigated. In B-NHL it is sufficient to associate CD19 , CD20 , CD79a and Ki-67 while in the t-NHL the CD2, CD3, CD4, CD8, CD20 shoud be generally used. The HL needs the addition of the CD30 to the basic panel while the multiple myeloma must be studied with CD138 and MUM1/IRF4.


Zini_piattaforme_di-diagnosi_in ematologia_Figura_1E Zini_piattaforme_di-diagnosi_in ematologia_Figura_2E Zini_piattaforme_di-diagnosi_in ematologia_Figura_3E

Fig.1E                                                         Fig.2E                                             Fig.3E

Zini_piattaforme_di-diagnosi_in ematologia_Figura_4E Zini_piattaforme_di-diagnosi_in ematologia_Figura_5E Zini_piattaforme_di-diagnosi_in ematologia_Figura_6E

Fig.4E                                                         Fig.5E                                             Fig.6E

Zini_piattaforme_di-diagnosi_in ematologia_Figura_7E Zini_piattaforme_di-diagnosi_in ematologia_Figura_8E Zini_piattaforme_di-diagnosi_in ematologia_Figura_9E

Fig.7E                                                         Fig.8E                                             Fig.9E

Zini_piattaforme_di-diagnosi_in ematologia_Figura_10E



Fig.1 MPDs ( prodromal phase): A) hypercellular BM in the PV panmyelosis. B) BM with enlarged megakaryocytes . C) Cluster of abnormal megkaryocytes in the PMF. HM Kvaniska et al. Am J Hematol 2010 – Fig.2 Megakaryocytes : CD61 staining (A) allows to identify more MGKs than H&E. BJ Wilkins J Clin Pathol 2011 –  Fig.3 Follicular Lymphoma. Arber et al. Am surg Pathol 2005 – Fig.4 Expression of MUM1: A) diffuse large B-cell lymphoma (DLBCL) with nuclear and cytoplasmic expression of MUM1( lymph node). B) DLBCL negative to MUM1, ( lymph node). C) strong expression of MUM1 in plasma cells.(BM trephine biopsy) B. Falini et al. Blood 2005 – Fig.5 Hodgkin lymphoma: Reed Sternberg cell(left). Reed Sternberg cell with lymphocytes and hystiocytes (in the middle). Reed Sternberg cells CD30 positive with nuclear stain of PAX-5(right). QY Zhang et al.Hematol Oncol Clin N Am 2009 – Fig.6 Hypocellular AML: A) clusters of immature cells . B) Blasts CD34 positive. JM Bennett et al. Hematologica 2009 – Fig.7 Mantle cell lymphoma: peripheral blood smear(up and left) and BM (bottom left) interstitial pattern in the trephine biopsy by CD20 stain (up and right) and cyclin D1( positive (bottom and right). QY Zhang et al. Hematol Oncol Clin N Am 2009 – Fig. 8 ALL: A) H&E. B) strong nuclear positività to PAX-5. C) cytoplasmic CD79a. D)Cytoplasmic and surface CD22 positivity. MR Nasr et al. Am J Clin Pathol 2010 – Fig.9 Sezary syndrome: cytological features Sezary cell (left), pattern of distribution by H&E (in the middle) and CD3 stain ( right). QY Zhang et al.Hematol Oncol Clin N Am 2009 – Fig.10 Multiple Myeloma. A)Plasma cells CD138 positive and B) Plasma cells CD138 negative. IB Bayer-Garner et al. Mod Pathol 2001




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