Blood film

Preparation

Two ways in preparing the peripheral blood films:

- smear

- by putting a drop on one side and spread it to another side, so look at the end side of the spread.

- several dead cells can be found due to the mechanical spreading

- monocytes, trombocytes aggregates are frequently found on the margin because they are large

- spinning

- by using a machine that 'spin'.

- equal spread

- but cells are 'blown', therefore less suitable for schistocytes (so if you see small spherocytes on spinning, think of having a smear to see schistocytes), for hairy cell leukemia (so if you see T lymphocytes with large cytoplasma, you should order smear).

For bone marrow

- the first aspiration is used

- always smeared directly after taken the sample

 

Stain

- May Grunwald Giemsa

- consisted of base (methylene blue) that stains acid (such as DNA in nucleus) and negative laden granules and acid (eosin, red) that stain base.

- blue glass (vs. normal red glass): can indicate paraprotein.

- we use Hematek machine that use Wright stain (modified Giemsa)

- Vital staining

- stain is mixed with the blood directly instead of after making a smear.

- RNA will appear as points instead of blue RBCs in May Grunwald.

- Special stain

- Myeloperoxidase (MPO): stains granulocytes and monocytes but not lymphocytes

- NCL: stains granulocytes but not lymphocytes and not monocytes

- Alpha naftyl: stains monocytes but not granulocytes and sometimes lymphocytes

- PAS: staining for granulocytes and sometimes lyphocytes
 

Some tips in recognizing cells

Myeloblasts

- Fine granules in the nucleus

- Light blue cytoplasma (vs. clear in monoytes or lymphocytes)

- Rounds in the nucelus (nucleolus)

- Blue cytoplasm

- Myeloblasts vs. lymphoblasts

- Myeloblasts: bilobular nucleus, red material in cytoplasma, larger

- Lymphoblats: large nucleus (fine granules vs. rough in lymphocytes), low cytoplasma (quite similar to lymphocytes), small

- With small expansion (like podocytes in lyphocytes): can be acute monoblastic leukemia

Promyelocytes - Myelocytes - Metamyelocytes - Bands

From earlier to later phase:

- less nucleolus in the nucleus (white)

- the cytoplasma become less dense

- the nucleus become more segmented (in metamyelocytes like it as Spanish guitar and in bands as dumbels)

- Promyelocyte vs. myeloblasts: primaire granules (red) in myeloblasts vs. none. Secondary granules: purple. Promyelocyte has significant nucleoli which look likes eyes.

Neutrophils

- granules

Eosinophils

- red granules

Basophils

- the nucleus is 'hidden' behind the granules

- the granules are larger than those of myelocytes

Lymphocytes

- dark nucleus

- large granular lymphocytes: empty cytoplasm but with red materials

Plasma cells

- exentric nucleus, very purple

- flame-like: in multiple myeloma (mostly IgD)

Monocytes

- very large

- granules or vacuoles (it has role in phagocytosis)

- in case of leukemia, monocytes can change the morphology, become large with empty cytoplasms

Erythroblasts

- Mickey mouse ears (pro erythroblasts)

- dark round nucleus (piknotic erythroblasts)
 

Changes in WBC or RBC noticed in special cases in peripheral blood

RBCs

- Basket cells (bite cells): G6PD deficiency

- Polychromasia: a lot of RNA, sign of erythropoesis

- Poikilocytosis: different forms, can appear in for example Thalassemia

- Malaria parasites in the RBCs can be ring-like or a cluster (gametocyte), look also at the gold coloured crystal hemozoine.

- Heinz body: small round inclusion in RBC, made of damaged hemoblobin in oxidant damage (i.e. thalassemia), not visible in Romanowsky stain. - Howell-Jolly bodies, basophilic (blue-) remnants of DNA in the damamaged spleen. - Pappenheimer body: iron granule; seen in sideroblastic anemia, sickle cell disease.

- teardrop: myelofibrosis (in combination with reactive lymphocytes).

Myeloblasts

Think of AML M3: bilobulaire nucleus, Auer rod, granular. The granula can come free during the treatment and can cause coagulation problem.

There are two types: microgranular, and hypergranular

Lymphoblasts

- Hand mirror: look like hand mirror, a variation of ALL

- Vacuolisation in lymphoblasts: Burkitt lymphoma or Burkitt leukemia

Erythroblasts

- Sieve-like nucleus and large erythroblasts: can be secondary to treatment using hydrea (used to treat essential thrombocytosis). This can give monocytair aspect of myelo-and metamyelocytes too.

Neutrophils granulocytes

- Hypersegmented neutrophils: Vitamine B12 deficiency anemia, medication (i.e. hydroxyurea for CML treatment)

- Hyposegmented (Pelger Huet): genetic, medication

- Pseudo Pelger Huet: myelodysplatic syndrome

- Bilobular neutrophil granulocytes: young female who smoke in large amount.

- Absence of granulocytes: myelodysplatic syndrome (in combination with pancytopenia or hypersegmentation)

- Large granulocytes: can be signs of maturity of neutrophils (after chemotherapy), this is in combination with the observation of the presence of blasts (can be up to 20%) and promyelocytes - myelocytes

- Auer Rod : Acute Myeloid Leukemia, Auer rod is always malignant, will not present in normal or reactive situation

Lymphocytes

- Atypical lymphocytes: large, much larger than red blood cells and purple 5only present in peripheral blood, not in marrow)

- Many large granular lymphocytes: transplantation, T-CLL

- 'brainlike cells': thinks of Sezary T-cell lymphoma

- When this atypical lymphocytes present in the context of CLL, think of prolymphocytes

- Vacuolisation in lymphocytes: lysososmal storage disease

Lymphocytes in Non Hodgkin lymphoma

- We can see the change in the peripheral blood (It is seldom that we can see the Hodgkin lymphocytes in the peripheral blood)

- Originated from lymph nodes

- Leukocytes > 5000/ mm3

- Mostly B-cells (nucleus more on peripheral)

- Vs. reactive lymphocytes: less blue cytoplasma, monoclonal (they look more similar)

- Vs. blasts: no granulocytes (blasts have red granulaocytes), less blue (blasts are quite blue)

- Several examples:

Chronic Lymphoid Leukemia

- in contradictory ot other leukemias, diagnosis can be performed from peripheral blood: football nucleus (blocked chromatin) of lymphocytes.

- A variation is prolymphocyte leukemia. These cells are quite similar to blasts, except they have larger nuceoli.

- They are not real leukemia (like CML which is myeloproliferative) but lyphoma. The name is derived by the invasion of the bone marrow.

Milt lymphoma

- hairy cell leukemia

- consider about this when in spin you see lymphocytes with large cytoplasm, then order the smear

- the hairy is seen only in smear but not in spin (because in spin the cells become pompous)

- no monocytes and leukepenia

- splenic villous leukemia

- look like hairy cells but the lymhocytes morphology is similar to normal lyphocytes next to the air

- plenty of leukocytes

Lymphoma originated from glands

- a wide variation in the morphology, we use the term atypical lyphoid cells in our laboratory

- in these cells already present in the peripheral blood, they are mostly already in adbanced stadia

- some descriptions:

- small no cytoplasma: normally not calculated, too similar

- hotdog: cleavage in the middle (quite similar to lymphoblasts) but they are smaller, no cytoplasma

- larger, more purple lymphocyte (blasts like but not blasts)

Megakaryocytes

- increased in number and in all stages: idiopathic thrombocytopenic purpura

- increased in number with one nucleus: MDS

- normal number and hypersegmented: essential trombocytosis

 

Blasts vs. lymphoma vs. atypical T lymphocytes

- Blast: fine chromatin, nucleoli, light blue, granular

- Lymphoma: empty cytoplasma

- Atypical T-lymphocytes: larger chromatin, more blue (purple if B-cells), larger cytoplasma (compared to blasts), more variation in size (especially in mononucleosis where you will have a lot of doubt awhen you are counting the cells, in mononucleosis the amount can be >17%)