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DNA Down Under

New section on RNA interference as a tool to block virus replication.

• When investigating the blood picture, its important to look at both the peripheral blood (PB) and bone marrow (BM) as the PB does not always mirror the BM.

• Anaemia - pallor, lethargy
• Leukaemic inhibition of haematopoiesis-crowding out)
• Ineffective erythropoiesis (eg. Dec. Vit. B12)
• Normocytic - Macrocytic normochromic anaemia at diagnosis
• White Cell Count (WCC) is not a good indicator, it may be Inc., Normal (N), or Dec. (more than half may not have an initial leukocytosis)
• Night sweats due to Inc. metabolism of the cancerous cells
  
• Neutropaenia - mouth, nose and eye infections
• Thrombocytopaenia - bruising - bleeding (may see megathrombocytes)
• Arthralgia (joint pain) in children because of pressured nerves ('bone pain")
• Splenomegaly - fullness, dragging sensation (50%)
• Hepatomegaly (25%)
• M5 - Gum and skin lesions - skin infiltration
• M3 - Bleeding can lead to disseminated intravascular coagulation (DIC)

1. Clinical data
2. Haematology profile
3. Examination of a stained blood film
4. Examination of a bone marrow aspirate
5. Cytochemistry and Tdt
6. Immunophenotyping
7. Cytogenetics
8. Electron microscopy
9. Gene rearrangements
10. Miscellaneous (tissue culture, oncogenes)

• The highest incidence of AL is found in individuals with chronic myeloproliferative/dysplastic diseases ("preleukaemia")
• Other "preleukaemic" diseases (considered stem cell disorders):
  PNH
  Aplastic Anaemia (AA)
  Multiple myeloma
  Lymphoma
  
• Blasts seen in leukaemias are neoplastic, therefore they may differ from normal in morphology and metabolism. They appear to develop with nuclear/cytoplasmic asynchrony (nucleus lags cytoplasm, cf IDA)
• Breakdown products of cell turnover (lactate dehydrogenase, uric acid) may increase markedly

• Rarely, the BM may appear hypoplastic, resembling an AA. Look for proportionate Inc. in blasts and fibroblasts (typical of AL, not AA)
• Consider leukaemia with a blood picture of Dec. Plt, Hb and weight loss, and look for Auer rods (Convalescence of granules. Not always seen, most often in M3. Immediate identification of an AML) when a large number of blasts are present.
• Erythrocyte Sedimentation Rate (ESR)
  • Affected by a number of factors, and as such is not considered diagnostically reliable without other data.
  • Factors include Red Blood Cell (RBC) shape (Dec. with spherocytes), number (Inc. with lower cell number) and size (Inc. with higher cell number)
  • Also plasma proteins (Inc. prolongs the ESR) and technical problems (how vertical the tube is, how dilute the specimen is and Inc. temp prolongs the ESR)
  • An ESR can reflect bacterial infection, rheumatoid arthritis, multiple myeloma and malignancy
• M0 ("m-naught") is included in the differential diagnosis of ALL
  • <3% are MPO POS
  • ALL markers are NEG
  • AML - CD13 and CD33 POS. Therefore classify this on the presence of myeloid markers not MPO
• Faggot Cells ("bundles" of rods) - Classical M3

Acute Myeloblastic Leukaemia (AML)

FAB	Category	Abbrevn.	Criteria
AML M1	Acute Myeloblastic Leukaemia	AML	15-20% of AML 30% or more of nonerythroid marrow cells are myeloblasts 3% of blasts or more stain for MPO (< in ALL) or Sudan Black Less than 10% promyelocytes +8 frequently seen
AML M2	Acute Myeloblastic Leukaemia with maturation	AML	30% of AML 30% or more of noneryhtroid marrow cells are myeloblasts >10% are promyelocytes <20% are monocytic t(8:21) is diagnostic
AML M3	Acute Promyelocytic Leukaemia	APL	5-10% of AML Most marrow cells are abnormal, hypergranular promeyelocytes Auer rods may be seen Classical-Hypergranulated, 80% leukopaenic Variant-Hyposegmented, leukoplasia, faggot cells Still an AL even though <30% blasts Granules contain procoagulants (thromboplastin-like) - can result in massive DIC if released with lysis due to cytotoxic drugs. Pretreat with prophylactic heparin t(15:17) is diagnostic
AML M4	Acute Myelomonocytic Leukaemia	AMML	15-20% of AML 30% or more of all nucleated marrow cells are blasts Granulocytes make up >20% of nonerythroid marrow cells. A monocytic component is identified by: _5x109/L or more monocytic elements in the blood and either: (1) 20% or more cells of monocytic lineage in the BM, or (2) A serum lysozyme level >3 ties normal _<5x109/L monocytic elements in the blood but 20% or more cells of monocytic lineage in the BM with cytochemical confirmation del/inv 16q
AML M5	Acute Monocytic Leukaemia	AMoL	15% of AML 30% or more of all nonerythroid marrow cells are monoblasts, promonocytes or monocytes Often associated with infiltration into gums/skin Weakness, bleeding and diffuse erythematous skin rash In M5A, 80% or more of all monocytic cells are monoblasts In M5B, <80% of monocytic cells are monoblasts (maturation) Poor prognosis t/del 11q
AML M6	Acute Erythroleukaemia	AEL	3-4% of AML 50% or more of all nucleated marrow cells are erythroid precursors, and 30% or more of the remaining nonerythroid cells are myeloblasts (if <30% then myelodysplasia) Often terminates in M1 or M2 Anaemia and striking aniso- or poikilocytosis, Howell Jolly bodies, ringed sideroblasts and megaloblastic changes (normal B12 and folate)
AML M7	Acute Megakaryoblastic Leukaemia	AMegL	2-4% of AML 30% or more of cells in the BM are of megakaryocytic lineage If marrow aspirate unobtainable (may need to trephine) due to fibrosis, diagnosis relies on the presence of; Excess blasts and maturing megakaryocytes as well as.. Circulating megakaryoblasts (confirm origin with platelet peroxidase + electron microscopy or MAb to vWF or glycoproteins Can occur de novo or as a progression from CGL/myelodysplasia Megakaryocytes can secrete a Plt-derived mitogenic factor capable of stimulating fibroblast proliferation

• Still give similar treatments for all AMLs
• M1, M2 & M3 have varying degrees of granulocytic differentiation
• M4 has granulocytic and monocytic differentiation
• Maturation may be indicative of a better prognosis

   

• 
  Figure 2. AML BM Examination Protocol

• 80% of leukaemias in 2-10 year olds - 10-20% of adults also so this group can not be disregarded in the diagnostic picture
• Prognosis is not as good for adults/infants/Ph' chromosome⁺/Increased blast counts as in children
• Neutropaenia, thrombocytopaenia (petechiae and ecchymoses more common than haemorrhage) and anaemia (similar to AML)
• Joint pain
• These blast cells have a tendancy to get into the CNS leading to meningeal leukaemia
• Higher incidence of splenomegaly and hepatomegaly. Also infiltrate into the lymphatics and bone marrow.
• In children there is often a prior viral infection, and the marrow may have been hypoplastic prior to leukaemia

 

	ALL
WCC	Inc. - Dec.
Plt.	Dec.
Leucocytosis	50%
Neutrpenia	Often marked
Anaemia	Normocytic, normochromic
BM	>30% lymphoblasts
Inclusions	No Auer rods
Morhology	No NRBCs or poikilocytes

Cytology	L-1 Best prognosis, most common ALL in children	L-2 Most frequent ALL in adults, similar inclusions to M2 AML	L-3 Worst ALL, morphology linked to B-cell ALL
	Lymphoblastic leukaemia with homogeneity	Lymphoblastic leukaemia with heterogeneity	Burkitt's-type lymphoblastic leukaemia
Cell size	Small	Large, heterogeneous	Large, homogeneous
Chromatin pattern	Homogensous within any one case	Heterogeneous within any one case	Homogeneous, stippled
Nuclear Shape	Smooth, occasional cleft	Irregular, clefts and indents	Smooth, oval-round
Nucleoli	Invisible-inconspicuous	1 or more	1 or more, prominent
Amount of cytoplasm	Little	Variable, often abundant	Moderately abundant
Cytoplasmic basophilia	Slight-moderate	Variable, sometimes deep	Very deep
Cytoplasmic vacuolization	Variable	Variable	Often prominent

 

• There is a link between immunological markers and prognosis but not morphology and prognosis.

	Cytochemistry				Imunophenotype
Type of ALL	MPO	PAS	ACP	NSE	Ia	Tdt	CD10	CD19	CD20	CD7	CD2
Pre-B	-	+	-	-	+	+	+	+	+	-	-
B Cell	-	-	-	-	+	+	-	+	+	-	-
Pre-T	-	+/-	+	+	-	+	-	-	-	+	+
T Cell	-	+/-	+	+	-	+	-	-	-	+	+

 

• Ia for B-lymphocytes
• Tdt is an ALL marker, CD10 only for Pre-B
• CD19 & CD20 for B-leukaemia types; CD2 & CD7 for T-leukaemia
• Differentiating T-leukaemia is not essential to patient outcome
• Correlation of FAB subtypes with immuntypes of ALL does not appear to have a clear pattern (except for L3 and B-ALL)
• Smear cells are often a sign of immaturity
• RBC are usually fairly normal in leukaemia, just too few
• Very few leukaemias have normal Hb