M5 can be classified into M5a (Acute Monoblastic Leukaemia without maturation) and M5b (Acute Monoblastic Leukaemia with maturation or Acute Monocytic Leukaemia). The subtyping of M5 depends solely on the morphologic examination of the marrow and the estimated % of monoblasts versus more mature monocytic cells, namely promoncytes and monocytes. The number of monoblasts in M5 should be >80%¹. Some haematologists commented that discrepancies may be observed because of individual's subjective interpretation of the cellular morphology. However, the difference in subtyping would not affect the clinical management of the disease. Auer rods in general are not present in M5a but are present in one-third of M5b².

1. Barbara J. Bain (1995), Blood Cells A Practical Guide, 2nd edition, p. 277, Blackwell Science.
2. Wintrobe's Clin Haem. 10th Ed. 1999; pp2215.

     Unlike classical cases of acute myeloid leukaemia, acute promyelocytic leukaemia (APL) usually presents with a low white cell count. Morphological diagnosis depends on recognition of the abnormal cells that resemble promyelocytes much more than myeloblasts. They have a lower nuclear to cytoplasmic ratio and granulated cytoplasm that sometimes contains a paranuclear hof. Yet they have a lobulated contour and occasional Auer rods, hence the term "abnormal promyelocytes" has been generally adopted to distinct them from the normal promyeloctyes.

     Hypergranulated forms with deep purplish cytoplasm and faggot cells (cells containing multiple Auer rods) are highly characteristics of APL. A microgranular variant cells with agranular or faintly granular cytoplasm is also recognized, which can easily be confused with monocytic leukaemia. Strong cytochemical staining for myeloperoxidase, Sudan Black B and chloroacetate esterase is a simple and quick test to support the diagnosis.

     APL is now routinely treated with the differentiating agent all-trans retinoic acid. The differentiating cells can be very abnormal morphologically. They may have irregular nuclei and hypogranulated cytoplasm, rendering it difficult if not impossible to classify them into various maturation stages by standard criteria. The term "intermediate cells" is used by some to describe these abnormal cells in circulation.

---------Contributed by Dr. C.C. SO (Haematologist, Department of Pathology, QEH)

HS0116 was taken from a patient undergone all-trans retinoic acid (ATRA) therapy for acute promyelocytic leukaemia (APL)[1]. The induction of maturation of the leukaemic clones attributed to the differentiating effect ATRA was generally noted in the myelocytes showing prominent granules and Auer rods.

APL comprises 5-10% of acute myeloblastic leukaemia (AML). Basically APL occurs in all walks of life; with male to female ratio being 2 to 1, the median age of 38 years but infrequent before 10 years of age. The most common presenting symptoms related to hemorrhagic disorders are bruisablility, bleeding gums, hemoptysis, epistaxis, petechiae, gastrointestinal bleeding and intra-cranial hemorrhage, whereas hepatosplenomegaly and lymphadenopathy may also present.

Haematologically white blood cell (WBC) count varies from marked leucopenia to marked leucocytosis. Patients with hypergranular APL often present with leucopenia of the median WBC count of 1.8 x 10⁹/L. On the contrary patients with the microgranular variant, AML-M3V, more often present with leucocytosis with the median WBC count of 83 x 10⁹/L. The majority of the patients are anaemic and thrombocytopenic: approximately 50% and 75% of the patients have haemoglobulin levels of less than 10g/dL and platelet counts being below 50 x 10⁹/L, respectively. Disseminated intravascular coagulation is evident in approximately 90% of the patients.

Morphologically APL is characterized by the presence of abnormal promyelocytes, which constitute more than 30% of the cells in the bone marrow. The nuclei are often bi-lobed or reniform and the cytoplasm is closely packed with large azurophilic granules. Numerous Auer rods characterized in bundles of faggots are randomly distributed within the cytoplasm. The use of the term “promyelocyte” in AML-M3 has created some confusion [2]. Despite the hypergranular features and the historical name of “promyelocytes”, these cells should be considered as blasts. In the bone marrow of patients with APL, the majority of non-erythroid cells are hypergranular blasts without significant neutrophilic maturation [3].

Hypergranular APL was initially the only recognized morphological subtype with the unique chromosomal translocation t(15;17)(q22;q21) demonstrated in the cytogenetic analysis [4]. However, cases of microgranular AML-M3 variant with t(15;17), that did not fulfil the usual AML-M3 criteria, were also reported [5].

The t(15;17) results the rearrangement of PML/rara chimeric transcripts. Genetically the retinoic acid receptor alpha gene, rara, on the chromosome 17q21 fuses with the zinc-finger-binding transcription domain of the promyelocytic leukaemia gene, PML, on the chromosome 15q22, giving rise to the PML/rara fusion gene product. In cases of APL with t(11;17)(q23;q21), the promyelocytic leukaemia zinc-finger gene, PLZF, is translocated to the rara gene on chromosome 17q21. Besides rearranged with PML and PLZF genes, the rara gene also fuses to the nucleophosmin gene, NPM, and the nuclear matrix associated gene, NuMA, leading to the formation of the reciprocal fusion proteins of N-RARa and RARa -N, respectively. This observation not only highlights the importance of retinoid metabolism, but also suggests that fusion partner genes with rara also play important roles in the epigenetics of APL [6]. Reverse transcription polymerase chain reaction for PML/rara mRNA can be used to monitor minimal residual disease of APL patients undergone chemotherapy.

References:

1. Huang M, Ye Y, Chen S, et al. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukaemia. Blood 1988; 72:567.
2. Bian BJ. Leukaemia Diagnosis. A Guide to the FAB Classification. Gower Medical Publishing. London. 1990.
3. Bennette JM, Catovsky D, Daniel MT et al. Proposal for the Classification of the acute leukaemia. Br J Haematol 1976; 33: 451.
4. Berger R, Bernheim A, Daniel MT, et al. t(15;17) translocation in acute promyelocytic leukaemia (M3) and cytological M3-variant. Nouv Rev Fr Haematol 1981; 23: 27.
5. Bennett JM, Catovsky D, Daniel MT et al. The French-American-British (FAB) Coorperative Group, Correspondence: a variant form of hypergranular promyelocytic leukaemia (M3). Br. J Haematol 1980; 44: 169.
6. Zelent A. Translocation of the RAR locus to the PML or PLZF gene in acute promyelocytic leukaemia. Br. J Haematol 1994; 86:451.

Blast crisis of CML [HS0216]

Natural history of chronic myeloid leukemia is characterized by 3 phases: chronic phase, accelerated phase and acute phase (blast crisis). All chronic myeloid leukaemias progress to an acute phase during the course of the disease, if left untreated. This acute phase may arise by transformation from the chronic phase, or an accelerated phase may precede it. The blasts are usually of myeloid lineage and can usually be identified by immunological markers and; less commonly (20%-30%) of lymphoid in origin, which may be PAS-positive and usually display the immunologic features of common ALL. Megakaryoblast or erythroblast transformation is characteristic but less frequent, and mixed lineage blast phases are somewhat more common. The laboratory features of the three phases are summarized in the following table:

 *  Pathological left-shift: appearance of immature granulocytopoietic forms at all stages with individual granulocytes showing qualitative changes such as anisocytosis, nuclear-cytolasmic asynchrony, and hyposegmentaion (pseudo-Pelger forms).

#   WHO definition.

WBC: granulocytopoiesis;      RBC: erythrocytopoieisis;     THP: thrombocytopoiesis       LAP: leukocyte alkaline phosphatase     Ph: chromosome: Philadelphia chromosome

Reference:

1. H. Loffler, J Rastetter. Atlas of Clinical Haematology, 5^th Edition. Springer. 2000. p142-3.

2. Wintrobe’s Clinical Haematology, 10^th Edition. 1999. p2343-2353

3. RL Bick. Haematology Clinical &Laboratory Practice.Mosby.1993. p1218

4. ES Jaffe, NL Harris, H Stein, JW Vardiman (Ed.)  Tumours of Haematopoietic and Lymphoid Tissue. WHO classification of Tumours. IARC 2001 p20–26.

Juvenile Chronic Myelomonocytic Leukaemia (JMML) [HS935]

Examination of the blood film of JMML is more helpful in the diagnosis of this disease than the bone marrow. The white cell count is only moderately raised, being about 50 x 10e9/L in most children, anaemia is common, and the blood film shows monocytes, abnormal granulocytes and some blasts. In contrast to chronic myelocytic leukaemia, platelet count is low. Neither Philadelphia chromosome with t(9;22)(q34;q11) is cytogenetically evident nor BCR/abl chimeric transcript is detected by reverse transcription-polymerase chain reaction. The great fascinations of JMML are the association of a raised level of fetal haemoglobin of greater than 10% and the spontaneous bone marrow culture without any supplement of the colony stimulating factor of granulocytes and macrophages.

Clinically JMML appears to be more common in boys and usually presents with pallor, splenomegaly and bleeding due to thrombocytopenia. The median survival of JMML children of age over 2 years at diagnosis was 6 months to one year whereas younger children had a median survival of over 4 years.

1. No RBC enlargement (to rule out P.vivax and P.ovale).
2. No Schuffner's dots seen when stained in pH7.2 Giemsa. (to rule out P.vivax and P.ovale).
3. Marginal forms / multiple infection / banana-shaped gametocytes not seen (P.falciparum unlikely).
4. Band form trophozoites in RBC (occasionally seen in HS816 suggestive of P. malariae).
5. Abundant golden iron pigments (suggestive of P.malariae).
6. Characteristic "daisy head" picture of 8 merozoites liberated from schizont (suggestive of P.malariae).

The term 'left shift' is a colloquial expression for a change in the cellular composition of the blood or marrow wherein cells are less mature than usual and is often misunderstood(1). An increase of younger forms (band form, metamyelocytes, myelocytes) suggests increased release of early myeloid cells from the marrow, which is often associated with acute infection and inflammation. However, this term is poorly defined and not readily quantifiable. It is necessary to establish the normal range of the average number of segments present in 100 neutrophils in individual laboratory before this term can be used to convey its true meaning(2). With the advent of automated cell counter, absolute level of the neutrophils is readily available and the presence of neutrophilia, cytoplasmic vacuolation, toxic granulation and Dohle bodies, etc. are sufficiently indicative for bacterial infection. We should therefore consider abandoning this obsolete term in reporting peripheral blood findings particularly in a QA exercise from the fourth survey of 1999.

Like left shift, this is an expression indicating greater maturity or that there is a shift towards an older cell type(1). This term should no longer be used for the same argument as above.

Reference:

1. Carol T. Kapff & James H. Jandl, Blood Atlas and Sourcebook of Haematology. Boston Little, Brown and Company 1981
2. G. Richard LEE et al. Wintrobe's Clinical Haematology. Vol 1. Ninth Edition Philadelphia, London,LEA & Febiger 1993

Last updated on 9 Nov., 2001.

Prepared by HKIMLSQAP Haematology & Serology Panel.

Copyright 2000 HKIMLSQAP. All Rights Reserved.