Plasma Cell Dyscrasia & Flow Cytometry in Hematology

Plasma Cell Dyscrasia

Normal Plasma Cell

• Eccentric nucleus—something like cartwheel chromatin
• The whitish area around the nucleus is called a Perinuclear Halo/hoff
• The area is white because there is a lot of Golgi apparatus present here.

Why do Plasma Cells Need Golgi Apparatus?

Plasma cells secrete immunoglobulin, proteins that, in turn, are formed by the Golgi apparatus.

Osteoblasts

• Similar to a plasma cell
• Eccentric nucleus on one side
• Paranucleur Halo/hoff

Osteoblast

• Present in bone
• Halo is away from the nucleus (para-nuclear).

Plasma Cell

• Present in bone
• Halo is perinuclear—just around the nucleus

Disorders

• Multiple myeloma
• Smouldering multiple myeloma
• Monoclonal gammopathy of undetermined significance (MGUS)
• Plasma cell leukemia
• Waldenstrom's macroglobulinemia
• Heavy chain disorder

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Multiple Myeloma: Genetics 

• t(11;14) (also seen in mantle cell lymphoma)
• Deletion 13q (also seen in CLL)
• Origin: Post-Germinal Centre B cell

Pathogenesis 

Increased Plasma cells

↓

Increased Ig (monoclonal)

↓

Produces Kappa or lambda light chains

↓

Causing light chain excess → Urine→Bence Jones Proteins

↓

Myeloma kidney is formed (A amyloid L light chain type)

↓

CKD with 20 hyperparathyroidism Increased calcium

• Normal Ig will produce (k & λ) light chains (60:40 ratio)
• Cast formed known as myeloma cast: fractured cast with breaks and cuts
• Benejones proteinuria: made up of a light chain
• IL 6 → Plasma cell proliferation → responsible for activating the RANKL pathway → osteoclastic activity → Bony Lytic Lesions (exam question)
• Plasma cells releasing DKK1 (DICKKOPF 1) block osteoblast production.
• No bone formation → Alkaline phosphatase level will be normal; otherwise, ALP level shoots up during bone formation.
• Calcium increases as it goes into the blood (since it is not utilized as no bone is formed)
• Elevation of calcium, but ALP is normal.

Clinical Features

• Elderly patients will have bone lytic lesions
• Skull: Salt and pepper (also seen in hyperparathyroidism)

• Vertebrae: low back pains

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Criteria

• Plasma cells >10%: in the bone marrow only, there is no plasma cell in blood vessels
• Ig > 3 gm/dL: Most common involved is IgG
• Myeloma defining event
  • C: calcium levels elevated (>11 gm/dL)
  • R: renal insufficiency (s.creatinine level > 2 mg/dL)
  • A- Anemia (Hb <10gm)
  • B- Bony lytic lesions
  • S: plasma cell >60%
  • Li: light chains ratio (involved /uninvolved) > 100
  • M- MRI at least 1 lesion of > 5 mm is seen

Multiple Myeloma Blood Findings

Rouleaux Formation

• RBCs are spread out & not sticking to each other because of a negative charge called Zeta potential
• Multiple myeloma will have lots of antibodies (immunoglobulin) covering RBCs Eliminating negative charges
• There is a loss of Zeta potential; RBCs will be stuck with each   forming rouleaux

Bone Marrow Aspirate Finding

• Russell Body: Intra cytoplasmic inclusion
• Dutcher Body: Intranuclear inclusion
• MOTT/mulberry cell
• Flame Cell: IgA-type multiple myeloma
• The composition of all these findings is Antibodies (Ig)

Bone Marrow Biopsy

• Eccentric nucleus, perinuclear halo
• Sheets of plasma cell

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Serum Protein Electrophoresis (Spep)

• Samples: blood/urine (antibodies are present in both)
• Normal blood sample is made up of Albumin and three-folded finger-like structure
  • Alpha 1 proteins (alpha 1 antitrypsin)
  • Alpha 2 protein (alpha 2 macroglobulin)
  • Beta (proteins)
  • Gamma antibodies (immunoglobulins)

Smouldering Multiple Myeloma: Criteria

• Plasma cell > 10%
• Ig > 3gm/dL
• There is no myeloma-defining event

Monoclonal Gammopathy of Undetermined Significance - Criteria (MGUS) 

• Plasma Cell <10 %
• Ig <3 gm/dL
• No MDE

Plasma Cell Leukaemia – Criteria 

• Plasma cells are present in bone marrow
• Plasma cells>20% of peripheral blood
• ALL and AML - Blast 20%

Waldenstrom's Macroglobulinemia (IgM)

• Also known as LPL (lymphoplasmacytic lymphoma)
• Cells: lymphocytes, plasma cells, and mast cells
• Genetics -
  • Deletion of chromosome 6q (most common)
  • MYD 88 mutation
• Clinical Features
  • IgM increase:
    • hyperviscosity:
      • Headache
      • Visual disturbance
      • Hemorrhage (cerebral, retinal)

Heavy Chain Disease 

• Alpha chain disease, Seligmann Disease
• Stomach - H. Pylori
• Small intestine - IPSID (Immunoproliferative Small Intestinal Disease.)

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MCQ's 

Q. The proliferation and survival of myeloma cells depend on which of the following cytokines?

1. IL-1
2. IL-6
3. IL-2
4. IL-5

Q. What are large, homogenous cytoplasmic inclusions in plasma cells called?

1. Dutcher bodies
2. Councilman bodies
3. Russell bodies
4. Mallory hyaline bodies

Q. What causes the M spike in Waldenstrom's macroglobulinemia?

1. IgM
2. IgG
3. IgA
4. IgD

Q. Is malignancy associated with Waldenstrom's macroglobulinemia?

1. Mycosis fungoides.
2. Smouldering myeloma
3. Primary effusion lymphoma
4. Lymphoplasmacytic lymphoma

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What is Flow Cytometry?

Flow cytometry is a FACS (fluorescence-activated cell sorting) machine.

Forward Scatter and Side Scatter

• When laser light is emitted, some of the light scatters forward (forward scatter FSC) and some scatters sideways (side scatter).
• If the cell is small, it scatters less light. If the cell is bigger, it scatters more light.
• Forward scatter indicates cell size; larger cells have greater forward scatter.
• Side scatter is proportional to cell granularity, where higher granularity results in higher side scatter.

1. Samples: Samples can be blood, bone marrow aspirate, FNAC, body fluids

2. Dyes: FITC (Fluoro Isothio Cyanate—green colored dye)

3. Hydrodynamic focusing: Sheath fluid is added to the sample to produce a single file of cells, which is known as hydrodynamic focusing. Sample + marker & dye + sheath fluid

4. Light: Blue-coloured laser light of wavelength 488 nm falls on the cells.

Lineage and Markers

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Questions:

Q. CD34+ /HLADR+ /MPO+ What is it?

Ans. AML

Q. CD34+ /TdT+ /CD3+/CD7+; What is it?

Ans. T-ALL

Q. Tdt+/ HLADR+/ CD10+/ CD19+; What is it?

Ans. B-ALL

Q. CD 34+/ HLADR+/ MPO+/CD 7+; What is it?

Ans. AML

Q. HLADR+/TdT+/ MPO+/CD19; What is it?

Ans. Biphenotypic Leukemia

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