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Morphological Diagnosis and Immunophenotypic Characterization of Onco-hematologic Malignancy (Acute Myelogenous Leukemia-M3 and M4): Two Pediatric Case Reports and Literature Review

Background: Acute myelogenous leukemia (AML) is an ensemble of malignant myeloid precursor bone marrow neoplasm. Acute promyelocytic leukemia (APL-M3) and acute myelomonocytic leukemia (AML-M4) are common sorts of pediatric AML. Even though these onco-hematologic malignancies are uncommon and sound for about 15% of childhood and adolescent leukemia. Case Report: We have reported two Sudanese female patients, a 9-years-old girl with APL-M3 and an 11-years-old with AML-M4. Interestingly, the patient with APL manifested bruises and coagulation disorders. Autoimmune hemolytic anemia (AIHA) and tonsillitis were associated with the AML-M4 patient. Conclusion: It is pertinent to conclude that the combined interpreted morphology and immunophenotyping are fundamental tools in the determination of acute leukemias. APL represents an emergency and the planning of diagnosis and executive management ought to be very short. AIHA is one of the possible causes of anemia in AML-M4 cases, if a blood transfusion is ordered, ought to be given consciously in such cases to remain away from unsafe hemolysis.

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Clinical Research in Hematology  •  Vol 3  •  Issue 1  •  2020 1
INTRODUCTION
A
cute myelogenous leukemia (AML) is the most
common family of leukemia. It is recognized
as a clonal hemopoietic stem cell disorder and
proclaimed by repression of differentiation resulting in the
gathering of cells at various stages of deficient maturation.
Furthermore, diminished production of mature hemopoietic
ingredient occurs.[1]
AML is rare in children and adolescents,
but most common in adults. It comprises 80% of acute
leukemias in adults and 15–20% in children.[2]
Acute
promyelocytic leukemia (APL) and acute myelomonocytic
leukemia are prevalent types of pediatric AML. The
frequency of APL among pediatric AML is less than 10%.
APL is a disease characterized by a discrimination block
at the promyelocytic stage. These unusual promyelocytes
are viewed as practically identical to blasts for the goal
of diagnosis. APL is linked to specific chromosomal
abnormalities (15;17) translocation, this translocation is
appropriate for diagnosis instead of blast count.[3]
AML-M4
is uncommon and appears approximately 3% of pediatric
AML. It is realized by 20% (WHO classification) or
30% (French-American-British [FAB] classification)
of myeloid and monocytoid cells in the peripheral blood
and bone marrow.[4]
Both APL-M3 and AML-M4 may be
associated with coagulation abnormalities.[3,5]
Diagnostic
techniques for both leukemias incorporate immature cell
count, morphology, cytochemistry, immunophenotyping,
cytogenetics, and histochemistry in connection with clinical
highlights. All these diagnostic techniques are integral.
Cell morphology remains the fundamental indicative
tool to survey the number and morphology of blast cells.
Immunophenotyping by flow cytometry is a strength
CASE REPORT
Morphological Diagnosis and Immunophenotypic
Characterization of Onco-hematologic Malignancy
(Acute Myelogenous Leukemia-M3 and M4): Two
Pediatric Case Reports and Literature Review
Bashir Abdrhman Bashir
Department of Hematology, Faculty of Medical Laboratory Sciences, Port Sudan Ahlia College, Sudan
ABSTRACT
Background: Acute myelogenous leukemia (AML) is an ensemble of malignant myeloid precursor bone marrow neoplasm.
Acute promyelocytic leukemia (APL-M3) and acute myelomonocytic leukemia (AML-M4) are common sorts of pediatric
AML. Even though these onco-hematologic malignancies are uncommon and sound for about 15% of childhood and
adolescent leukemia. Case Report: We have reported two Sudanese female patients, a 9-years-old girl with APL-M3 and an
11-years-old with AML-M4. Interestingly, the patient with APL manifested bruises and coagulation disorders. Autoimmune
hemolytic anemia (AIHA) and tonsillitis were associated with the AML-M4 patient. Conclusion: It is pertinent to conclude
that the combined interpreted morphology and immunophenotyping are fundamental tools in the determination of acute
leukemias. APL represents an emergency and the planning of diagnosis and executive management ought to be very short.
AIHA is one of the possible causes of anemia in AML-M4 cases, if a blood transfusion is ordered, ought to be given
consciously in such cases to remain away from unsafe hemolysis.
Key words: Acute myeloid leukemia, acute non-lymphoblastic leukemia, acute myelogenous leukemia-M4, APL, Sudan
Address for correspondence:
Dr. Bashir Abdrhman Bashir Mohammed, Department of Hematology, Faculty of Medical Laboratory Sciences, Port
Sudan Ahlia College, Port Sudan, Sudan.
https://doi.org/10.33309/2639-8354.030101 www.asclepiusopen.com
© 2020 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license.
Bashir: Morphology and immunophenotypic of AML-M3 and M4
2 Clinical Research in Hematology  •  Vol 3  •  Issue 1  •  2020
adjuvant tool in portraying cell surface and cytoplasmic
markers in AML-M3 and AML-M4. The exhibition of
characteristic myeloid descent markers CD34, CD13,
CD33, CD117, CD11b, CD11c, CD14, and human leukocyte
antigen–D-related (HLA-DR) permits the distinction of
both leukemias to form other sorts of AML.[6]
CASE REPORT
Case 1
A9-years-old Sudanese girl presented with fever, generalized
weakness, easy fatigability, extensive bruising, petechiae
located in upper and lower limbs, and occasional epistaxis.
She was in a generally unsatisfactory condition. She had
not had hepatosplenomegaly and lymphadenopathy. The
vital signs were seen normal, heart rate 132/bm, blood
pressure 105/73 mmHg, and oxygen saturation 96%.
The complete blood count revealed slight normocytic
normochromic anemia, marked leukocytosis, and extreme
thrombocytopenia. The peripheral blood film indicated that
88% of leukocytes are neoplastic promyelocytes (faggot
cells) [Figure 1]. Her coagulogram manifested prothrombin
time 20 s, control 13.5 s, international normalization ratio 1.4,
partial thromboplastin time 43 s, control 25–40 s, fibrinogen
was normal, and elevated d-dimer [Table 1].
Bone marrow aspiration indicated hypercellular marrow
appearing propagation of blasts. The blasts were medium to
huge in size with high N:C proportion, moderate quantities
of cytoplasm, fine nuclear chromatin, and 1–2 noticeable
nucleoli.Afewoftheblastswerebinucleated.Megakaryocytes
were slightly reduced in number, but typical in morphology,
erythroid-myeloid ratio diminish, and some mitotic shapes
are frequently detected. Cytochemical myeloperoxidase and
Sudan Black B were positive and periodic acid-Schiff was
negative. The flow cytometry examination for bone marrow
aspiration demonstrated blasts/immature cells positive for
CD45, CD13, CD33, and CD117 in the blasts, and negative
for CD11b, CD34, and HLA-DR as well as increased side
scattered [Figure 2 and Table 1]. The final determination in
view of morphological and immunophenotypic findings was
indicated acute promyelocytic leukemia (APL), FAB-M3
hypergranular was resolved. Unfortunately, the patient died
before therapy initiation.
Case 2
An 11-years-old Sudanese girl presented with a history of
marked fatigue, fever, and severe pain in the throat. There
was no other contributory history. She did not take explicit
prescriptions or received blood items before investigation.
On examination, she had a high-grade fever and congested
both tonsils. The soft palate was also inflamed. The vital
signs reveal heart rate 126/bm, blood pressure 100/70,
body mass index 18.6, and O2
saturation 99%. She had
no previous clinical proof of infection. A throat swab for
culture and sensitivity was ordered and Streptococcus
sensitive to Amoxyclav was reported. Complete
blood check at presentation demonstrated normocytic
normochromic anemia, elevated leukocyte count, and
severe thrombocytopenia. The peripheral blood smear
indicated some spherocytes. The differential leukocyte
count exhibited 40.3% blasts and 35% monocytic series
[Figure 3]. Cytochemical periodic acid-Schiff was negative,
myeloperoxidase and non-specific esterase were positive.
The presentation of spherocytes is evidence of autoimmune;
therefore, a direct anti-human globulin test was obtained
positive. The sort of antibody could be recognized as
immunoglobulin G with monospecific antibodies. The
reticulocyte check was just 0.7%. Nonappearance of
reticulocytosis was clarified by bone marrow invasion by
neoplastic blast cells. Virology screening for hepatitis C
virus, hepatitis B virus, and HIV was negative. The finding
of acute myelomonocytic leukemia M4 was affirmed by
immunophenotyping on bone marrow aspirate which
uncovered 45% myeloblast population positive for CD34,
CD13, CD33, CD45, CD117, and HLA-DR and the
monocytoid series were positive for CD14, CD64, CD11c,
CD11b, and HLA-DR [Figure 4 and Table 2].
The patient began dexamethasone 10 mg each 12 h with
continuous improvement of hemoglobin level as it arrived
at 10.3 g/dl when the chemotherapy began once more.
The patient finished the primary cycle and begun steroid
withdrawal with a stable hemoglobin level. The hemoglobin
dropped again consequently to chemotherapy triggered
myelosuppression. The assessment of the primary pattern
of chemotherapy with bone marrow aspiration appeared
complete recovery. The Coombs’ test was remained positive
after the first dose and reported negative after the second
Figure 1: Peripheral blood picture; (a) Faggot cell and blast with blebs; (b and d) blast cells with many Auer rods; (c) Blast with
intracytoplasmic microgranules, Auer rod, and internuclear bridge
a b c d
Bashir: Morphology and immunophenotypic of AML-M3 and M4
Clinical Research in Hematology  •  Vol 3  •  Issue 1  •  2020 3
Test Patient result Reference interval Interpretation
Hematology
WBC 36.20 4.0–10.0 × 109
/l Critical
RBC 3.33 3.8–5.5 × 1012
/l L
Hemoglobin 10.3 12–15 g/dl L
Hematocrit 27 35–45% L
MCV 81 78–98 fl N
MCH 31 26–33 N
MCHC 38 30–36 N
RDW 13 11.5–15% N
Platelet count 21 150–400 × 109
/l L
MPV 10.3 9–13 fl N
PDW 17.1 13–17 fl Critical
P-LCR 24.6 13–43% N
Absolute differential counts
Neutrophils 1.2 1.5-6.0 × 109
/l L
Lymphocytes 0.4 6.0-8.3 × 109
/l L
Monocytes 0 0.1-1.3 × 109
/l L
Eosinophils 0 0-0.5 × 109
/l N
Blasts 1.0 0 × 109
/l Critical
Promyelocytes 31.9 0 × 109
/l Critical
Myelocytes 0.7 0 × 109
/l Critical
Metamyelocytes 1.0 0 × 109
/l Critical
Frequency differential counts
Band neutrophils 0 0–5% N
Segmented neutrophils 3 20–40% L
Lymphocytes 1 40–70% L
Atypical lymphocytes 0 0–6% N
Monocytes 0 0–10% N
Eosinophils 0 0–6% N
Basophils 0 0–1% N
Metamyelocytes 3 0% Critical
Myelocytes 2 0% Critical
Promyelocytes 88 0% Critical
Blasts 3 0% Critical
Normoblasts 2 100 N
Coagulation
PT 20 11–16 s H
INR 1.4
PTT 43 25–40 s H
Fibrinogen 3.63 2–4 g/dl N
D-Dimer 7.0  0.3 mg/l Critical
Table 1: Laboratory findings for patient with AML-M3
(Contd...)
Bashir: Morphology and immunophenotypic of AML-M3 and M4
4 Clinical Research in Hematology  •  Vol 3  •  Issue 1  •  2020
Test Patient result Reference interval Interpretation
Immunophenotyping
Variables Approximately positive (%) Interpretation
CD45 85.0 Positive
CD13 55.4 Positive
CD33 78.5 Positive
CD117 82.0 Positive
CD34 11.5 Negative
HLA-DR 16.25 Negative
CD11b 22.1 Negative
WBC: White blood cells, L: Low, RBC: Red blood cells, MCV: Mean corpuscular volume, N: Normal, MCH: Mean corpuscular hemoglobin,
MCHC: Mean cell hemoglobin concentration, RDW: Red blood cell distribution width, H: High, MPV: Mean platelet volume, PT: Prothrombin
time, INR: International Normalized Ratio, PTT: Partial thromboplastin time, CD: Cluster of differentiation, HLA-DR: Human leukocyte
antigen–D-related. AML: Acute myelogenous leukemia
Table 1:(Continued)
Figure 2: Scattergram of CD45 versus side scatter (SS)
indicate the granularity of leukemic cells (black arrow)
Figure 4: Scattergram of CD45 versus side scatter (SS)
indicates the myeloid population in (black arrow) and
monocytic series in (green)
short course of dexamethasone. At present, the patient is
steady in remission with acceptable hemoglobin levels with
no proof of hemolysis.
Regrettably, clonal chromosomal variations could not be
investigated in either patient because short of facilities.
DISCUSSION
AMLis an onco-hematologic neoplastic disease characterized
by the proliferation of myeloid cell antecedents with or
without maturation. The underlying symptoms of AML are
connected to anemia, neutropenia, and thrombocytopenia,
which promote due to bone marrow infiltration of neoplastic
blasts.[7]
AMLcan detect at any age category, but the incidence
increases with age.[2]
The AML in childhood comprised
20% up to 27% compared to adult AML comprised of 76%
up to 86%.[8]
For over 20 years, the FAB order for intense
leukemia has been a significant framework of classification.
This order empowered the diagnosis of an assortment of
morphologic and cytochemical subtypes of intense leukemia
through an organized rule. Morphological studies alongside
with cytochemical stains consolidated the diagnosis of over
Figure 3: Blood smear; (a) monocytic series (monoblast,
promonocytes, and monocyte); (b) blasts with prominent
nucleoli and monocytic series; (c) spherocytes noted in a
different area of the blood film (arrow)
c
b
a
Bashir: Morphology and immunophenotypic of AML-M3 and M4
Clinical Research in Hematology  •  Vol 3  •  Issue 1  •  2020 5
Test Patient result Reference interval Interpretation
Hematology
WBC 84.0 4.0–10.0 × 109
/l Critical
RBC 2.44 3.8–5.5 × 1012
/l L
Hemoglobin 9.6 12–15 g/dl L
Hematocrit 24 35–45% L
MCV 99 78–98 fl N
MCH 40 26–33 N
MCHC 39 30–36 H
RDW 16.4 11.5–15% H
Platelet count 42 150–400 × 109
/l L
MPV 10.3 9–13 fl N
PDW 17.1 13–17 fl H
P-LCR 24.6 13–43% N
Absolute differential counts
Neutrophils 1 1.5–6.0 × 109
/l L
Lymphocytes 6.8 6.0–8.3 × 109
/l N
Monocytes 10 0.1–1.3 × 109
/l H
Eosinophils 0.7 0–0.5 × 109
/l H
Blasts 40.3 0 × 109
/l Critical
Promonocytes 25.2 0 × 109
/l Critical
Myelocytes 0 0 × 109
/l Critical
Metamyelocytes 0 0 × 109
/l Critical
Frequency differential counts
Band neutrophils 0 0–5% N
Segmented neutrophils 1.2 20–40% L
Lymphocytes 8 40–70% L
Atypical lymphocytes 0 0–6% N
Monocytes 12 0–10% N
Eosinophils 0.8 0–6% N
Basophils 0 0–1% N
Metamyelocytes 0 0% Critical
Myelocytes 0 0% Critical
Promonocytes 30 0% Critical
Blasts 48 0% Critical
Normoblasts 0 100 N
Coagulation
PT 15.5 11–16 s N
INR 1.0
PTT 36 25–40 s N
Fibrinogen 2.9 2–4 g/dl N
D-Dimer 0.2 0.3 mg/l N
Table 2: Laboratory outcomes for a patient with AML-M4
(Contd...)
Bashir: Morphology and immunophenotypic of AML-M3 and M4
6 Clinical Research in Hematology  •  Vol 3  •  Issue 1  •  2020
Test Patient result Reference interval Interpretation
Immunophenotyping
Variables Approximately positive (%) Interpretation
CD45 62.0 Positive
CD13 70.4 Positive
CD33 87.6 Positive
CD117 82.0 Positive
CD34 49.5 Positive
HLA-DR 75.8 Positive
CH11c 76.4 Positive
CD11b 58.0 Positive
CD14 94.0 Positive
CD64 79.1 Positive
WBC: White blood cells, L: Low, RBC: Red blood cells, MCV: Mean corpuscular volume, N: Normal, MCH: Mean corpuscular hemoglobin,
MCHC: Mean cell hemoglobin concentration, RDW: Red blood cell distribution width, H: High, MPV: Mean platelet volume, PT: Prothrombin
time, INR: International Normalized Ratio, PTT: Partial thromboplastin time, CD: Cluster of differentiation, HLA-DR: Human leukocyte
antigen–D-related. AML: Acute myelogenous leukemia
Table 2:(Continued)
80% of AML cases.[9]
To the best of our knowledge, pediatric
AML has been reported in the literature.
Both of our patients fulfilled the criteria of the presence of
40% blasts in the peripheral blood smear in two cases. In
both of our patients, practically the entirety of the cell could
be recognized as blast cell, which made up over half of the
nucleated marrow cells. We herein, reported both of the cases
as FAB M3 and M4 subtypes, as there was more than 40%
differentiation in the myeloid lineage as well as the erythroid
antecedents were scant.
IntheAPL-M3,theblastdemonstratedabnormalintracytoplasmic
granules, the nuclei are bilobed or have a monocytoid view.
APL with t(15;17) (q22;q12) is an AML in which abnormal
promyelocytes predominate. Both hypergranular (“typical”
APL) and hypogranular (microgranular) presentations can be
observed. Three other morphological subcategories of APL-M3
were accounted for:APL-M3 with basophilic morphology,APL-
M3 with M2-like blast morphology, and APL-M3 with M1-like
blast morphology (early promyelocytic).[3]
Morphologically,
most pediatrics APL saw as hypergranular APL, along with
significant coagulopathy with serious bleeding episodes that
enhanced outcomes.[10]
In case 1, the coagulopathy findings,
morphological diagnosis, and cytochemical were clearly
observed the AML-M3. Immunophenotypically, the triple-
negative of (CD34-, HLA-DR-, CD11b-) alongside with
positive (CD33+, CD13+, CD117+) was proof of the diagnosis
of APL-M3.
Acute myelomonocytic leukemia represents 5–10% of
the entirety of AML. It is characterized by a significantly
augmented leukocyte along with the presence of myeloid and
monocytoid series in peripheral blood and/or bone marrow.[11]
Monocytic series (monoblasts and promonocytes) comprise
at least 20% of all marrow blood cells. The monoblasts are
vast with plentiful cytoplasm containing few granules and
pseudopodia. The nucleus is vast and immature and may
have multiple nucleoli. Promonocytes also are present and
may have contorted nuclei resembling (embryo shape).
Acute myelomonocytic leukemia with abnormal eosinophil
(AMML Eo) has also been recognized in the medical
literature.[11]
In case 2, present a patient of de novo AML-M4
associated with autoimmune hemolytic anemia (AIHA). The
case is managed with antibiotics, steroids, and chemotherapy.
The patient highlighted remarkable improvement for anemia
after the steroid doses with declines in all clinical and
laboratory findings of hemolysis when the patient went into
abatement.A similar picture of AML-M4 with autoantibodies
against erythrocytes with positive anti-human globulin test
with the clue of hemolysis has been reported by Essa et al.[12]
The method of advancement of AIHA in de novo AML-M4 is
muddled. A few reports hypothesize that an immune response
was delivered against a tumor antigen expressed on malignant
erythroblast derived erythrocytes. Nonetheless, this was
precluded in some cases because antibodies were likewise
directed toward the intact erythrocytes. Another clarification
might be identified with the advancement of immunologic
regulatory cells as suppressor T cells, which permitted the
advancement of autoimmunity.[12]
Eventually, morphological
performance, positive myeloperoxidase, and alpha naphthyl
acetate (non-specific esterase) indicated for AML-M4 and
confirmed by positive immunophenotyping CD34, CD13,
CD33, CD45, CD117, and HLA-DR for myeloid series and
CD14, CD64, CD11c, CD11b, and HLA-DR for monocytoid
series.

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Morphological Diagnosis and Immunophenotypic Characterization of Onco-hematologic Malignancy (Acute Myelogenous Leukemia-M3 and M4): Two Pediatric Case Reports and Literature Review

  • 1. Clinical Research in Hematology  •  Vol 3  •  Issue 1  •  2020 1 INTRODUCTION A cute myelogenous leukemia (AML) is the most common family of leukemia. It is recognized as a clonal hemopoietic stem cell disorder and proclaimed by repression of differentiation resulting in the gathering of cells at various stages of deficient maturation. Furthermore, diminished production of mature hemopoietic ingredient occurs.[1] AML is rare in children and adolescents, but most common in adults. It comprises 80% of acute leukemias in adults and 15–20% in children.[2] Acute promyelocytic leukemia (APL) and acute myelomonocytic leukemia are prevalent types of pediatric AML. The frequency of APL among pediatric AML is less than 10%. APL is a disease characterized by a discrimination block at the promyelocytic stage. These unusual promyelocytes are viewed as practically identical to blasts for the goal of diagnosis. APL is linked to specific chromosomal abnormalities (15;17) translocation, this translocation is appropriate for diagnosis instead of blast count.[3] AML-M4 is uncommon and appears approximately 3% of pediatric AML. It is realized by 20% (WHO classification) or 30% (French-American-British [FAB] classification) of myeloid and monocytoid cells in the peripheral blood and bone marrow.[4] Both APL-M3 and AML-M4 may be associated with coagulation abnormalities.[3,5] Diagnostic techniques for both leukemias incorporate immature cell count, morphology, cytochemistry, immunophenotyping, cytogenetics, and histochemistry in connection with clinical highlights. All these diagnostic techniques are integral. Cell morphology remains the fundamental indicative tool to survey the number and morphology of blast cells. Immunophenotyping by flow cytometry is a strength CASE REPORT Morphological Diagnosis and Immunophenotypic Characterization of Onco-hematologic Malignancy (Acute Myelogenous Leukemia-M3 and M4): Two Pediatric Case Reports and Literature Review Bashir Abdrhman Bashir Department of Hematology, Faculty of Medical Laboratory Sciences, Port Sudan Ahlia College, Sudan ABSTRACT Background: Acute myelogenous leukemia (AML) is an ensemble of malignant myeloid precursor bone marrow neoplasm. Acute promyelocytic leukemia (APL-M3) and acute myelomonocytic leukemia (AML-M4) are common sorts of pediatric AML. Even though these onco-hematologic malignancies are uncommon and sound for about 15% of childhood and adolescent leukemia. Case Report: We have reported two Sudanese female patients, a 9-years-old girl with APL-M3 and an 11-years-old with AML-M4. Interestingly, the patient with APL manifested bruises and coagulation disorders. Autoimmune hemolytic anemia (AIHA) and tonsillitis were associated with the AML-M4 patient. Conclusion: It is pertinent to conclude that the combined interpreted morphology and immunophenotyping are fundamental tools in the determination of acute leukemias. APL represents an emergency and the planning of diagnosis and executive management ought to be very short. AIHA is one of the possible causes of anemia in AML-M4 cases, if a blood transfusion is ordered, ought to be given consciously in such cases to remain away from unsafe hemolysis. Key words: Acute myeloid leukemia, acute non-lymphoblastic leukemia, acute myelogenous leukemia-M4, APL, Sudan Address for correspondence: Dr. Bashir Abdrhman Bashir Mohammed, Department of Hematology, Faculty of Medical Laboratory Sciences, Port Sudan Ahlia College, Port Sudan, Sudan. https://doi.org/10.33309/2639-8354.030101 www.asclepiusopen.com © 2020 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license.
  • 2. Bashir: Morphology and immunophenotypic of AML-M3 and M4 2 Clinical Research in Hematology  •  Vol 3  •  Issue 1  •  2020 adjuvant tool in portraying cell surface and cytoplasmic markers in AML-M3 and AML-M4. The exhibition of characteristic myeloid descent markers CD34, CD13, CD33, CD117, CD11b, CD11c, CD14, and human leukocyte antigen–D-related (HLA-DR) permits the distinction of both leukemias to form other sorts of AML.[6] CASE REPORT Case 1 A9-years-old Sudanese girl presented with fever, generalized weakness, easy fatigability, extensive bruising, petechiae located in upper and lower limbs, and occasional epistaxis. She was in a generally unsatisfactory condition. She had not had hepatosplenomegaly and lymphadenopathy. The vital signs were seen normal, heart rate 132/bm, blood pressure 105/73 mmHg, and oxygen saturation 96%. The complete blood count revealed slight normocytic normochromic anemia, marked leukocytosis, and extreme thrombocytopenia. The peripheral blood film indicated that 88% of leukocytes are neoplastic promyelocytes (faggot cells) [Figure 1]. Her coagulogram manifested prothrombin time 20 s, control 13.5 s, international normalization ratio 1.4, partial thromboplastin time 43 s, control 25–40 s, fibrinogen was normal, and elevated d-dimer [Table 1]. Bone marrow aspiration indicated hypercellular marrow appearing propagation of blasts. The blasts were medium to huge in size with high N:C proportion, moderate quantities of cytoplasm, fine nuclear chromatin, and 1–2 noticeable nucleoli.Afewoftheblastswerebinucleated.Megakaryocytes were slightly reduced in number, but typical in morphology, erythroid-myeloid ratio diminish, and some mitotic shapes are frequently detected. Cytochemical myeloperoxidase and Sudan Black B were positive and periodic acid-Schiff was negative. The flow cytometry examination for bone marrow aspiration demonstrated blasts/immature cells positive for CD45, CD13, CD33, and CD117 in the blasts, and negative for CD11b, CD34, and HLA-DR as well as increased side scattered [Figure 2 and Table 1]. The final determination in view of morphological and immunophenotypic findings was indicated acute promyelocytic leukemia (APL), FAB-M3 hypergranular was resolved. Unfortunately, the patient died before therapy initiation. Case 2 An 11-years-old Sudanese girl presented with a history of marked fatigue, fever, and severe pain in the throat. There was no other contributory history. She did not take explicit prescriptions or received blood items before investigation. On examination, she had a high-grade fever and congested both tonsils. The soft palate was also inflamed. The vital signs reveal heart rate 126/bm, blood pressure 100/70, body mass index 18.6, and O2 saturation 99%. She had no previous clinical proof of infection. A throat swab for culture and sensitivity was ordered and Streptococcus sensitive to Amoxyclav was reported. Complete blood check at presentation demonstrated normocytic normochromic anemia, elevated leukocyte count, and severe thrombocytopenia. The peripheral blood smear indicated some spherocytes. The differential leukocyte count exhibited 40.3% blasts and 35% monocytic series [Figure 3]. Cytochemical periodic acid-Schiff was negative, myeloperoxidase and non-specific esterase were positive. The presentation of spherocytes is evidence of autoimmune; therefore, a direct anti-human globulin test was obtained positive. The sort of antibody could be recognized as immunoglobulin G with monospecific antibodies. The reticulocyte check was just 0.7%. Nonappearance of reticulocytosis was clarified by bone marrow invasion by neoplastic blast cells. Virology screening for hepatitis C virus, hepatitis B virus, and HIV was negative. The finding of acute myelomonocytic leukemia M4 was affirmed by immunophenotyping on bone marrow aspirate which uncovered 45% myeloblast population positive for CD34, CD13, CD33, CD45, CD117, and HLA-DR and the monocytoid series were positive for CD14, CD64, CD11c, CD11b, and HLA-DR [Figure 4 and Table 2]. The patient began dexamethasone 10 mg each 12 h with continuous improvement of hemoglobin level as it arrived at 10.3 g/dl when the chemotherapy began once more. The patient finished the primary cycle and begun steroid withdrawal with a stable hemoglobin level. The hemoglobin dropped again consequently to chemotherapy triggered myelosuppression. The assessment of the primary pattern of chemotherapy with bone marrow aspiration appeared complete recovery. The Coombs’ test was remained positive after the first dose and reported negative after the second Figure 1: Peripheral blood picture; (a) Faggot cell and blast with blebs; (b and d) blast cells with many Auer rods; (c) Blast with intracytoplasmic microgranules, Auer rod, and internuclear bridge a b c d
  • 3. Bashir: Morphology and immunophenotypic of AML-M3 and M4 Clinical Research in Hematology  •  Vol 3  •  Issue 1  •  2020 3 Test Patient result Reference interval Interpretation Hematology WBC 36.20 4.0–10.0 × 109 /l Critical RBC 3.33 3.8–5.5 × 1012 /l L Hemoglobin 10.3 12–15 g/dl L Hematocrit 27 35–45% L MCV 81 78–98 fl N MCH 31 26–33 N MCHC 38 30–36 N RDW 13 11.5–15% N Platelet count 21 150–400 × 109 /l L MPV 10.3 9–13 fl N PDW 17.1 13–17 fl Critical P-LCR 24.6 13–43% N Absolute differential counts Neutrophils 1.2 1.5-6.0 × 109 /l L Lymphocytes 0.4 6.0-8.3 × 109 /l L Monocytes 0 0.1-1.3 × 109 /l L Eosinophils 0 0-0.5 × 109 /l N Blasts 1.0 0 × 109 /l Critical Promyelocytes 31.9 0 × 109 /l Critical Myelocytes 0.7 0 × 109 /l Critical Metamyelocytes 1.0 0 × 109 /l Critical Frequency differential counts Band neutrophils 0 0–5% N Segmented neutrophils 3 20–40% L Lymphocytes 1 40–70% L Atypical lymphocytes 0 0–6% N Monocytes 0 0–10% N Eosinophils 0 0–6% N Basophils 0 0–1% N Metamyelocytes 3 0% Critical Myelocytes 2 0% Critical Promyelocytes 88 0% Critical Blasts 3 0% Critical Normoblasts 2 100 N Coagulation PT 20 11–16 s H INR 1.4 PTT 43 25–40 s H Fibrinogen 3.63 2–4 g/dl N D-Dimer 7.0 0.3 mg/l Critical Table 1: Laboratory findings for patient with AML-M3 (Contd...)
  • 4. Bashir: Morphology and immunophenotypic of AML-M3 and M4 4 Clinical Research in Hematology  •  Vol 3  •  Issue 1  •  2020 Test Patient result Reference interval Interpretation Immunophenotyping Variables Approximately positive (%) Interpretation CD45 85.0 Positive CD13 55.4 Positive CD33 78.5 Positive CD117 82.0 Positive CD34 11.5 Negative HLA-DR 16.25 Negative CD11b 22.1 Negative WBC: White blood cells, L: Low, RBC: Red blood cells, MCV: Mean corpuscular volume, N: Normal, MCH: Mean corpuscular hemoglobin, MCHC: Mean cell hemoglobin concentration, RDW: Red blood cell distribution width, H: High, MPV: Mean platelet volume, PT: Prothrombin time, INR: International Normalized Ratio, PTT: Partial thromboplastin time, CD: Cluster of differentiation, HLA-DR: Human leukocyte antigen–D-related. AML: Acute myelogenous leukemia Table 1:(Continued) Figure 2: Scattergram of CD45 versus side scatter (SS) indicate the granularity of leukemic cells (black arrow) Figure 4: Scattergram of CD45 versus side scatter (SS) indicates the myeloid population in (black arrow) and monocytic series in (green) short course of dexamethasone. At present, the patient is steady in remission with acceptable hemoglobin levels with no proof of hemolysis. Regrettably, clonal chromosomal variations could not be investigated in either patient because short of facilities. DISCUSSION AMLis an onco-hematologic neoplastic disease characterized by the proliferation of myeloid cell antecedents with or without maturation. The underlying symptoms of AML are connected to anemia, neutropenia, and thrombocytopenia, which promote due to bone marrow infiltration of neoplastic blasts.[7] AMLcan detect at any age category, but the incidence increases with age.[2] The AML in childhood comprised 20% up to 27% compared to adult AML comprised of 76% up to 86%.[8] For over 20 years, the FAB order for intense leukemia has been a significant framework of classification. This order empowered the diagnosis of an assortment of morphologic and cytochemical subtypes of intense leukemia through an organized rule. Morphological studies alongside with cytochemical stains consolidated the diagnosis of over Figure 3: Blood smear; (a) monocytic series (monoblast, promonocytes, and monocyte); (b) blasts with prominent nucleoli and monocytic series; (c) spherocytes noted in a different area of the blood film (arrow) c b a
  • 5. Bashir: Morphology and immunophenotypic of AML-M3 and M4 Clinical Research in Hematology  •  Vol 3  •  Issue 1  •  2020 5 Test Patient result Reference interval Interpretation Hematology WBC 84.0 4.0–10.0 × 109 /l Critical RBC 2.44 3.8–5.5 × 1012 /l L Hemoglobin 9.6 12–15 g/dl L Hematocrit 24 35–45% L MCV 99 78–98 fl N MCH 40 26–33 N MCHC 39 30–36 H RDW 16.4 11.5–15% H Platelet count 42 150–400 × 109 /l L MPV 10.3 9–13 fl N PDW 17.1 13–17 fl H P-LCR 24.6 13–43% N Absolute differential counts Neutrophils 1 1.5–6.0 × 109 /l L Lymphocytes 6.8 6.0–8.3 × 109 /l N Monocytes 10 0.1–1.3 × 109 /l H Eosinophils 0.7 0–0.5 × 109 /l H Blasts 40.3 0 × 109 /l Critical Promonocytes 25.2 0 × 109 /l Critical Myelocytes 0 0 × 109 /l Critical Metamyelocytes 0 0 × 109 /l Critical Frequency differential counts Band neutrophils 0 0–5% N Segmented neutrophils 1.2 20–40% L Lymphocytes 8 40–70% L Atypical lymphocytes 0 0–6% N Monocytes 12 0–10% N Eosinophils 0.8 0–6% N Basophils 0 0–1% N Metamyelocytes 0 0% Critical Myelocytes 0 0% Critical Promonocytes 30 0% Critical Blasts 48 0% Critical Normoblasts 0 100 N Coagulation PT 15.5 11–16 s N INR 1.0 PTT 36 25–40 s N Fibrinogen 2.9 2–4 g/dl N D-Dimer 0.2 0.3 mg/l N Table 2: Laboratory outcomes for a patient with AML-M4 (Contd...)
  • 6. Bashir: Morphology and immunophenotypic of AML-M3 and M4 6 Clinical Research in Hematology  •  Vol 3  •  Issue 1  •  2020 Test Patient result Reference interval Interpretation Immunophenotyping Variables Approximately positive (%) Interpretation CD45 62.0 Positive CD13 70.4 Positive CD33 87.6 Positive CD117 82.0 Positive CD34 49.5 Positive HLA-DR 75.8 Positive CH11c 76.4 Positive CD11b 58.0 Positive CD14 94.0 Positive CD64 79.1 Positive WBC: White blood cells, L: Low, RBC: Red blood cells, MCV: Mean corpuscular volume, N: Normal, MCH: Mean corpuscular hemoglobin, MCHC: Mean cell hemoglobin concentration, RDW: Red blood cell distribution width, H: High, MPV: Mean platelet volume, PT: Prothrombin time, INR: International Normalized Ratio, PTT: Partial thromboplastin time, CD: Cluster of differentiation, HLA-DR: Human leukocyte antigen–D-related. AML: Acute myelogenous leukemia Table 2:(Continued) 80% of AML cases.[9] To the best of our knowledge, pediatric AML has been reported in the literature. Both of our patients fulfilled the criteria of the presence of 40% blasts in the peripheral blood smear in two cases. In both of our patients, practically the entirety of the cell could be recognized as blast cell, which made up over half of the nucleated marrow cells. We herein, reported both of the cases as FAB M3 and M4 subtypes, as there was more than 40% differentiation in the myeloid lineage as well as the erythroid antecedents were scant. IntheAPL-M3,theblastdemonstratedabnormalintracytoplasmic granules, the nuclei are bilobed or have a monocytoid view. APL with t(15;17) (q22;q12) is an AML in which abnormal promyelocytes predominate. Both hypergranular (“typical” APL) and hypogranular (microgranular) presentations can be observed. Three other morphological subcategories of APL-M3 were accounted for:APL-M3 with basophilic morphology,APL- M3 with M2-like blast morphology, and APL-M3 with M1-like blast morphology (early promyelocytic).[3] Morphologically, most pediatrics APL saw as hypergranular APL, along with significant coagulopathy with serious bleeding episodes that enhanced outcomes.[10] In case 1, the coagulopathy findings, morphological diagnosis, and cytochemical were clearly observed the AML-M3. Immunophenotypically, the triple- negative of (CD34-, HLA-DR-, CD11b-) alongside with positive (CD33+, CD13+, CD117+) was proof of the diagnosis of APL-M3. Acute myelomonocytic leukemia represents 5–10% of the entirety of AML. It is characterized by a significantly augmented leukocyte along with the presence of myeloid and monocytoid series in peripheral blood and/or bone marrow.[11] Monocytic series (monoblasts and promonocytes) comprise at least 20% of all marrow blood cells. The monoblasts are vast with plentiful cytoplasm containing few granules and pseudopodia. The nucleus is vast and immature and may have multiple nucleoli. Promonocytes also are present and may have contorted nuclei resembling (embryo shape). Acute myelomonocytic leukemia with abnormal eosinophil (AMML Eo) has also been recognized in the medical literature.[11] In case 2, present a patient of de novo AML-M4 associated with autoimmune hemolytic anemia (AIHA). The case is managed with antibiotics, steroids, and chemotherapy. The patient highlighted remarkable improvement for anemia after the steroid doses with declines in all clinical and laboratory findings of hemolysis when the patient went into abatement.A similar picture of AML-M4 with autoantibodies against erythrocytes with positive anti-human globulin test with the clue of hemolysis has been reported by Essa et al.[12] The method of advancement of AIHA in de novo AML-M4 is muddled. A few reports hypothesize that an immune response was delivered against a tumor antigen expressed on malignant erythroblast derived erythrocytes. Nonetheless, this was precluded in some cases because antibodies were likewise directed toward the intact erythrocytes. Another clarification might be identified with the advancement of immunologic regulatory cells as suppressor T cells, which permitted the advancement of autoimmunity.[12] Eventually, morphological performance, positive myeloperoxidase, and alpha naphthyl acetate (non-specific esterase) indicated for AML-M4 and confirmed by positive immunophenotyping CD34, CD13, CD33, CD45, CD117, and HLA-DR for myeloid series and CD14, CD64, CD11c, CD11b, and HLA-DR for monocytoid series.
  • 7. Bashir: Morphology and immunophenotypic of AML-M3 and M4 Clinical Research in Hematology  •  Vol 3  •  Issue 1  •  2020 7 The limitation of this report is that molecular analysis and cytogenetics were not used parallel with the cases. CONCLUSION Our discoveries propose integral utilization of immunophenotyping with morphology improves outcomes in the determination of acute leukemias. Further examination with the utilization of the expanded panel of monoclonal antibodies is suggested. Moreover, the advancement of reciprocal diagnostic methods such as cytogenetics and molecular studies ought to be produced for the additional exact conclusion of AML and its subtypes. APL represents an emergency and the planning of diagnosis and executive management ought to be very short. AIHA is one of the possible causes of anemia in AML-M4 cases, if a blood transfusion is ordered, ought to be given consciously in such cases to remain away from unsafe hemolysis. REFERENCES 1. Rubnitz JE, Gibson B, Smith FO. Acute myeloid leukaemia. Hematol Oncol Clin N Am 2010;24:35-63. 2. Osman IM, HumeidaAA, Eltayeb O,Abdelrhman I, Elhadi TA. Flowcytometric immunophenotypic characterization of acute myeloid leukemia (AML) in Sudan. Int J Hematol Dis 2015;2:10-7. 3. D’Angelo G, Ceriani L, Hotz AM, Mazzola D, Ranalli R. Acute promyelocytic leukemia in four-year-old female: A case report. Adv Lab Med Int 2012;2:111-8. 4. An Q, Fan CH, Xu SM. Recent perspectives of pediatric leukemia-an update. Eur Rev Med Pharmacol Sci 2017;21:31-6. 5. Dicke C, Amirkhosravi A, Spath B, Jiménez-Alcázar M, Fuchs T, Davila M, et al. Tissue factor-dependent and- independent pathways of systemic coagulation activation in acute myeloid leukemia: A single-center cohort study. Exp Hematol Oncol 2015;4:22. 6. Peters JM, Ansari MQ. Multiparameter flow cytometry in the diagnosis and management of acute leukemia. Arch Pathol Lab Med 2011;135:44-54. 7. Ferrara F, Schiffer CA. Acute myeloid leukaemia in adults. Lancet 2013;381:484-95. 8. Ahmad SQ, Yusuf R, Burney S. Frequency of various types of leukaemias diagnosed at PAF hospital Mianwali. Pak Arm Forces Med J 2015;65:474-7. 9. Belurkar S, Mantravadi H, Manohar C, KurienA. Correlation of morphologic and cytochemical diagnosis with flowcytometric analysis in acute leukemia. J Cancr Res Ther 2013;9:71. 10. Aksu T, Fettah A, Bozkaya IO, Bastemur M, Kara A, Çulha VK, et al. Acute promyelocytic leukemia in children: A single center experience for turkey. Mediterr J Haematol Infect Dis 2018;10:e2018045. 11. Baviskar JB. Incidence of acute and chronic leukemias in rural area at tertiary care teaching hospital: A five years of study. Ind J Pathol Oncol 2016;3:710-3. 12. Essa N, El-Ashwah S, Denewer M, Essam Y, Mabed M. Autoimmune hemolytic anemia in a patient with acute myelomonocytic leukemia. J Blood Disord Transfus 2016;7:336. How to cite this article: Bashir BA. Morphological Diagnosis and Immunophenotypic Characterization of Onco-hematologic Malignancy (Acute Myelogenous Leukemia-M3 M4): Two Pediatric Case Reports and Literature Review. Clin Res Hematol 2020;3(1):1-7.