Clinical Hematology

Chapter Page
1. Introduction 1
2. Manual CBC 3
- WBCs 3
- Platelets count 3
- PCV or Hct 4
- Retics count 5
- Hb Estimation 6
3. RBCs morphology 7
4. WBCs morphology 12
5. BM examination 14
6. Examples 18

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 Clinical Hematology

Introduction
- EDTA:
1. Types:
 K2 EDTA – Na2 EDTA → cholate Ca which is essential for coagulation.
2. Dose: 1.5 mg/1ml blood
3. Preparation: 6gm powder → 100 ml distal water.
4. Use: in CBC
5. Cases:
 Excess EDTA:
- RBCs shrinkage (↓ PCV, ↑MCHC).
- Platelets → swells → fragments.
- WBCs → degeneration
 Inadequate:
- RBCs shrinkage (↓ PCV, ↑MCHC).
- Platelets → swells → fragments.
- WBCs → degeneration
- Citrate
1. Na3 citrate: forms non ionizable salts of Ca.
2. Preparation → 32 gm/L.
3. In PT,PTT → 9:1 (blood : citrate)
4. In ESR → 4 : 1
5. In blood group, cross matching
- Heparin
1. Dose: 10-20 IU/L
2. Preparation: antithrombin (‫)مسحة هيبارين‬
3. In osmotic fragility, tissue typing and separation of mononuclear cell layer.
4. Not used in: CBC → faint blue background & clumping of WBCs.

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 Clinical Hematology
- Effect of blood storage:
a. Quantitative changes:
1. ↑ PCV, MCV, Osmotic fragility & PT
2. ↓ ESR, platelets & RBCs.
b. Qualitative changes:
1. RBCs > 6 hours → spherocytosis
2. WBCs → ragged, ill defined, cytoplasm, vacuoles, irregular.
3. Lobulation of nucleus (of neutrophil, monocytes and lymphocytes).
Note:
That direct film without anticoagulant in diagnosis of (lead toxicity – platelets aggregation
disorders).

PT
- ISI: curve between test thromboplastin and reference thromboplastin.
- INR: patient PT/controlled PT of ISI of the kit

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 Clinical Hematology

Manual CBC
A. WBCs
1. Preparation: 380 micron acetic acid + 20 micron blood → mix for 5 minutes →
fill the haemocytometer.
2. WBCs count = No of cells counted 4 squares x dilution / volume of 4 squares
= Number of cells counted x 50 / microL.

B. Platelets count
1. 380 micron ammonium oxalate + 20 micron blood → Mix → wait 10-15 minutes → fill
the haemocytometer.
2. Then put haemocytometer in humid chamber (pertidish + wet filter paper).
3. Wait 20 minutes → count the refractile dots = platelets in 5 small squares.
4. Platelets count = N0 of cells in 5 small squares x dilution / volume of 5 small squares.
= N0 of cells counted x 1000 → in modified Nubeir haemocytometer
= N0 of cells counted x 800 → in Nubeir haemocytometer
Notes:
- Count platelets from blood film = we count 10 fields x 2000 / microL.
- Platelets should be counted manually and automated if
1. marked microcytosis (thalessemia)
2. If many shictocytes because RBCs are small enough to be counted as platelets in
automation → false ↑ platelets count.
3. If giant platelets (Bernard Soulier syndrome) → platelets are large enough to be counted
as RBCs → false ↓ platelets count.
- Ammonium oxalate:
1. Should be filtered – Should be discard if any turbid or fungus growth.
2. Kept at 4 C˚ - Prepare small amount (500 ml at a time).

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 Clinical Hematology

C. PCV or Hct
1. Definition: volume occupied by RBCs expressed as a fraction of whole blood volume.
2. Value:
- Anemia screening
- Calculation of blood indices
- Calibration of automated blood count system.
3. Methods:
a. Macro-method by wintrabe tube.
b. Micro-hematocrit (which used now)
c. In automated → PCV is calculated not measured.
4. Principle: packing of RBCs by centrifugation of whole blood in capillary tube at high
(relative centrifugal force).
5. Centrifugation: at force = 12,000 g for 3-5 minutes
6. Abnormalities:
a. ↓ PCV:
- True: anemia
- False: excess EDTA → shrinkage of RBCs.
b. ↑ PCV:
- True: polycythemia, haemconcentration.
- False: ↑ plasma trapped in between RBCs e.g. sickle cell, other causes of poikilocytosis
7. Capillary tube:
- Plain or heparin coated
- And leaving 1.5 cm unfilled → seal by plastic seal or heating by a fine flame →
centrifuge with the sealed end → laterally → read on a special scale.
8. Notes: normally → trapped plasma in between RBCs = 2-3 %, ↑ in sickle cell = 20%.

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 Clinical Hematology

D. Retics count
1. Definition: Immature RBCs containing remnants of ribosomal RNA.
2. Principle: basic dye (Brillien Cresyl blue or supravital stain) + Ribosomal
RNA (acid) → precipitation of blue granules or filaments.
3. Method:
- Equal volume of blood + dye (e.g. 2 drops) → mix + close by stopper → incubates 20
minutes at 37C˚ → gentle mix and spread a film.
4. Automated:
- By florescent material or flow cytometry.
5. Count:
Retics % = number of retics counted x 100 / number of total RBCs counted.
= number/5 = the percentage (0.5 – 2.5 %).
6. Notes:
- If anemia → pseudoreticulocytosis → corrected retics = retics x Hct of patient / normal Hct
- Normal Hct = 45.
- RPI = Corrected Retics x maturation time.
- Retics stain (BCB):
 2 drops of blood after mixing + 2 drops of BCB → incubate 20 minutes at 37 C˚ in water
bath → then spread a film.
 In anemia → ↑ angel of spreader > 45C˚
 In polycythemia → ↓ angel of spreader > 45C˚
- Leishman stain:
 Cover the slide with the dye for 3 minutes.
 Dilute by drops of distal water + jet of air by using pipette.
 Leave for 15 minutes on the bar.
 Rinse with tap water + leave to dry.

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 Clinical Hematology

E. Hb Estimation
The measurement done by photometric or colorimetric methods:
1. Cyanmet-Hb method. 3. Oxy-Hb method.
2. Alkaline haematin method 4. Sahli's acid haematin method
 Cyanmet-Hb method
 Principle: Hb K-ferricyanide Met-Hb K-cyanide cyanmet-Hb (yellow colour).
 Measured by a spectrophotometer at wave length 540 mm
 Drabkin solution (K-ferricyanide, K-cyanide, Buffer, non-ionic detergent H2O).
 Features of Drabkin: (‫)مهم‬
- Clear – pale yellow – PH = 7 – 7.4
- Stored at room temperature – In brown glass bottle (not exposed to light).
 We measure against water as blank at 540 mm absorbance must be zero.
 Method: 5ml drabkin + 20 micron blood → mix → wait for 5 minutes before reading against
distal water (at 540) then against drabkin.
 Hb concentration = sample absorbance x standard concentration / standard absorbance.
Or = sample absorbance x factor.
 Special cases: false ↑ results due to turbidity by:
- ↑ TLC (CML) → centrifuge and take supernatant.
- Lipemia → plasma blank – ↑ Plasma protein (MM).
 Note: Hb standard
- Hb standard = solution of hemoglobin cyanide (haemotrol) → (in the form of 10 ml
ampoule concentration = 600 mg/liter) → written on ampoule.
.drabkin ‫ من ال‬serial dilution ‫ ← نعمل‬Hb curve ‫عند عمل ال‬ -
- Then the standard calculated by:
1. Concentration written on ampoule x dilatation = 15 gm/dL.
2. Read the absorbance of each dilution and blot the curve.
3. Hb curve → straight linear
4. Should be done for each newly prepared drabkin and to calibrate the photometer.
5. Concentration of the test sample can be obtained from the curve.

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 Clinical Hematology

RBCs morphology

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 Clinical Hematology

The comment should be on:

1. Size 2. Shape 3. Staining 4. Inclusion 5. Arrangement
6. Immature forms 7. Haemoparasite
1. Size
- Normally → normocytic = same size of nucleus of small lymphocyte
- Microcyte (If RBCs size < than lymphocyte)
1. Iron deficiency anemia 5. Thalassemia
2. Sideroblastic anemia 6. Lead poisoning
3. Atransferrinemia 7. Unstable Hb
4. Anemia of chronic diseases

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 Clinical Hematology
- Macrocyte (If RBCs size > than lymphocyte)
a. Megaloblastic
1. Vit-B12 deficiency 3. Congenital dyserythropoietic anemia
2. Folate deficiency
b. Non-megaloblastic
1. Liver diseases 3. Alcoholism 5. Post splenectomy 7. MDS
2. Myxedema 4. Pregnancy 6. Aplastic anemia 8. Neonates

2. Shape
- Normally biconcave shape.
- Abnormalities:
a) Spherocyte:
1. Hereditary: spherocytosis
2. Acquired:
- Autoimmune hemolytic anemia
- ABO incompatible
- Post transfusion
- Stored blood sample
- All types of hemolytic anemia except (pyruvate kinase, Rh, PNH & unstable-Hb).

a) Ovalocytes and Elliptocyte:

- Hereditary ovalocytosis or elliprocytosis if > 90% of the cells.
- Megaloblastic anemia
- Microcytic hypochromic anemia
- MF
- Liver disease
b) Target cell: Cell surface > volume
- Chronic liver disease , cholestatic jaundice
- Thalessemia major
- Hb-C (other Hbopthy = Hb-SS, Hb-AC, Hb-EE, Sickle B-thalessemia).
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 Clinical Hematology
- Splenectomy
c) Sickle cell:
- Sickle cell anemia (Hb-SS) (+ target cells ± Howel jolly bodies).
- Sickle cell trait (Hb-A + Hb-S) → sickling in hypoxia only.
- Sickle B-thalessemia (+ Microcytic hypochromic anemia + HSM)\
Note: A Target cell is a feature of sickle cell diseases.
d) Tear drop cell:
- Myelofibrosis - Hemolytic anemia
- Thalessemia - MDS
e) Others:
 Crenate RBCs: ‫خليا مشرشره من بره‬
- Stored blood sample
- Bad anticoagulant
- Uremia
- Cardiopulmonary by pass.
 Acanthocyte: ↑ number of projections
- Chronic liver disease.
- Post splenectomy.
 Burr cells: one or few spines.
- Uremia
 Stomatocyte:
- Artifact - Chronic liver disease
- Alcoholism - Thalessemia
- Megaloblastic anemia - Iron deficiency anemia
- Mechanical stress: DIC – TTP – HUS – Microangiopathic hemolytic anaemia
 Schistocytes (helmet = fragmented RBCs).

3. Staining
- Normally pink colour, central pallor > 1/3 size of the cell.
- Hypochromasia:

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 Clinical Hematology
 Central pallor markedly > 1/3 size of the cell.
 All causes of Microcytic hypochromic anemia.
- Anisochromasia
 Dimorphic picture (some cells hypochromic and others normochromic).
 Causes:
1. Sideroblastic anemia
2. Combined deficiency (↓ B12 and ↓ iron).
3. Blood transfusion to patient with hypochromic anemia
4. Iron deficiency anemia responding to iron therapy.

4. Inclusion
 Howel jolly bodies (nuclear remnant)
- Megaloblastic anemia
- Post splenectomy
- Splenic atrophy (autosplenectomy)
- Unstable Hb.
 Basophilic stippling (punctuate basophilic): -ve iron stain
- Lead poisoning - Thalessemia
- Megaloblastic anemia - Liver disease
- Unstable Hb - Infection

 Papperheimer bodies (minute black granules single or pairs): +ve iron stain
- Post splenectomy
- Haemoserdrosis
- Hemolytic anemia
 Nucleus: Normoblastic cells.

5. Arrangement
- Roleaux (pile of coins):
 Due to ↑ ESR (see causes of ↑ ESR in RBCs book).
- Autoagglutination:

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 Clinical Hematology
 Due to autoantibodies (autoimmune hemolytic anemia).

6. Immature forms
Normobastaemia
- Post hemorrhagic
- Post hemolytic crisis
- Post splenectomy
- Thalassemia major
- BM infiltration (leucoerythroblastic anemia, leukemia, lymphoma, MF …….etc).
Notes:
1. Normoblast + sever anisopokilocytosis = Thalassemia.
2. Normoblast + spherocytes + autoagglutination = AIHA
3 Corrected TLC = TLC x 100 / 100 + normoblast

7. Haemoparasite
- Malaria ringed form
- Schizont
- Gametocyte

WBCs morphology
- Myeloid series
 Blasts
 Promyelocyte
 Myelocyte
 Metamyelocyte
 Band
 Shift to left: band > 5% or promyelo, myelo, meta in peripheral blood.
 Shift to right: hypersegmented neutrophil > 5 segments = megaloblastic anemia.
 Segmented neutrophil
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 Clinical Hematology
- Granulocytes
 Neutrophil
 Esinophils
 Basophils
- Lymphoid series
 Blast (no Auer rods)
 Prolymphocyte (‫)عين الكتكوت‬
 Lymphocyte small
- Monocytes
- Esinophils
- Basophils
Some Neutrophil Abnormalities
1. Granules
- Toxic granulation:
 Increase number and density
 In infection and inflammation ….etc.
- Hypogranular: in CML, AML and MDS.
2. Vacuoles
 Unstained phagosomes
 In sever sepsis and stored blood sample.
3. Bacteria: free in cytoplasm or in vacuoles → over whelming sepsis.
4. Pelger Huet: bilobed neutrophil → congenital, MDS, CML and AML.
5. Pyknotic: dead neutrophil with dense featureless nuclei → sever sepsis and stored blood.
Important notes:
1. Activated lymphocyte = immunoblast = turk cell = virocyte → convert to
plasma cell: caused by → B-lymphocyte (viral, bacterial & autoimmune).
2. Atypical lymphocyte:
-Large size, eccentric nucleus, more abundant cytoplasm, rim of basophilia, pale blue
cytoplasm, fine azurophilic granules, pseudopodia tends to adhere to adjacent RBCs, ±
nucleolus.

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 Clinical Hematology
-Causes: viral infection (EBV & CMV), CLL, lymphoma, chronic infection (Toxo & TB).

BM examination
a. In the low power: we should comment on
1. Cellularity
2. megakaryocytes
3. infiltration (Gaucher – Niman pick, neuroblastoma)
b. By oil immersion lens
→ BM counting:
1. Myeloid
2. Erythroid
3. Lymphocytes
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 Clinical Hematology
4. Plasma cells
5. Blasts
We have to comment in 10 items in BM report:
1. BM Cellularity: hypo or hypercellular
2. M/E ratio: normally 3:1 or 4:1
3. Myeloid series:
4. Erythroid series
5. Lymphocytes: Normally up to 20% of ANCs
6. Plasma cells : Normally up to 10% of ANCs
7. Blasts
8. Megakaryocytes
9. No Haemoparasite (Malaria or leishmania).
10.No abnormal or malignant infiltration at examined site.

Details
1. BM Cellularity: hypo or hypercellular
- Ratio between haemopoeitic cells are 75% and fat cells are 25%.
- If cells > fat: hypercellular BM
- If fat > cells: hypocellular BM
2. M/E ratio: normally 3:1 or 4:1
- If there is Erythroid hyperplasia:
a. Megaloblastic anemia (most common slide):
 Prominence of early forms (early normoblasts).
 Arrest of maturation, Large cell size & Delayed nuclear maturation
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 Clinical Hematology
 Stippling appearance of nucleus
 N/C asynchrony → nuclear maturation
 Increase mitotic figures → Howel jolly bodies.
 Giant meta and stab.
b. Iron deficiency anemia (very rare slide now)
 Predominance of late normoblast.
 Delayed Hbinization with ragged cytoplasm.
3. Myeloid series:
- Activity: active, hyperactive or depressed.
- Maturity: predominant cells, maturation arrest or normal, mitotic figure, sequence of
maturation …..etc.
- Abnormality: Pelger Huet, hypogranulation, hypersegmentation, toxic granulation.
4. Erythroid series:
5. Lymphocytes: Normally up to 20% of ANCs
- In CLL & lymphoma → infiltration of BM ≥ 30% lymphocytes of ANCs, either small
mature looking lymphocytes or immature.
6. Plasma cells: Normally up to 10% of ANCs
- In MM: ↑ plasma cells ≥ 10% of ANCs together with myeloma cells
- In plasma cell leukemia & Waldenstrom → ↑ number of plasma cells in BM.
- Reactive plasmacytosis → chronic liver & collagen diseases, aplastic anemia, malignancy.
7. Blasts
- In acute leukemia → ↑ blast cells ≥ 20% of ANCs.
8. Megakaryocytes
- Normally: 1-2/ lower power field
- Mostly juvenile or mature.
- Platelet separation: adequate or inadequate.
- Dysmegakaryopoiesis: MDS, megaloblastic anemia & MPD.
- Comment:
 Adequately seen
 Depressed

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 Clinical Hematology
 Decreased
 Increased
 Frequently seen or infrequently seen.
9. No Haemoparasite (Malaria or leishmania).
10.No abnormal or malignant infiltration at examined site: with abnormal sheets of
cells → haemopoeitic or non-haemopoeitic.

Examples
1. General Scheme for differential count
We make this table twice in the exam:
 1st one film the comment of the WBCs
 2nd one form the film we count and prepare it.
Cell Differential Absolute x 109 /L Normal rang x 109 /L

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- Neutrophil - 2–7
- Stab (band) - Up to 5%
- Lymphocyte - 1–4
- Atypical - Up to 5%
- Monocyte - Up to 1
- Esinophil - Up to o.5
- Basophil - Up to 0.1
- Meta
- Myelo
- Promyelo
- Blast
- Normoblast / WBCs

Remember in exam we must do corrected TLC = TLCx 100 / 100+normoblast.

Normoblast in peripheral blood: Sever hemorrhage, post-splenectomy & hemolytic anemia
(thalassemia - AIHA)
2. General Scheme for BM counting
Myeloid Erythroid Lymphocyte Plasma cells Blast cells
- MPD: - Anemia -LPD - MM - Acute leukemia
 CML  Megaloblastic CLL ≥ 20 % of ANCs
 ET  Iron Def. Lymphoma
- M3 promyeloblast - MDS -Atypical
- Leukomoid reaction - M6 lymphocytes

Remember that we have to comment in 10 items in BM report:

Case 1: MPD most probably CML

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 Clinical Hematology

1. BM Cellularity & M/E ratio: is markedly hypercellular with ↑ M/E ratio 20:1.
3. Myeloid series: is markedly hyperactive, all stages of maturation of granulocytic series
are present with ↑ number of basophilic, esinophilic elements.
4. Erythroid series: is relatively depressed 5. Lymphocytes: constitute 3 % of ANCs
6. Plasma cells : constitute < 1% of ANCs 7. Blasts: constitute 4% of ANCs
8. Megakaryocytes: are increased both juvenile, mature with adequate platelets separation,
occasional dwarf megakaryocytes are seen.
9. No Haemoparasite could be detected.
10. No other abnormal or malignant infiltration at examined site.
Conclusion:
BM is markedly hypercellular with markedly hyperactive myeloid series suggesting a case of
MPD most probably CML to be confirmed by NAP score (↓ in CML even zero) & cytogenetic
→ +ve t (9: 22).

Case 2: A case of acute leukemia

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 Clinical Hematology

1. BM Cellularity: is markedly hypercellular

2. M/E ratio: normally 3:1 or 4:1
3. Myeloid series 4. Erythroid series: 5. Megakaryocytes are depressed
6. Lymphocytes: 7. Plasma cells: ……….. % of ANCs
8. Blasts: infiltration of BM by about 80% blasts, characterized by: open chromatin,
prominent nucleolus and abundant cytoplasm (in AML).
9. No Haemoparasite.
10. No other abnormal or malignant infiltration at examined site.
Conclusion: BM is markedly hypercellular and infiltrated by about 80% blast cells → a case
of acute leukemia for further investigations:
1. Cytochemical stain (MPO): +ve AML – -ve ALL – Mo, M7.
2. IPT 3. Cytogenetic 4. Molecular technique 5. E/M ratio

Case 3: A case of multiple myeloma

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 Clinical Hematology

1. BM Cellularity: markedly hypercellular

2. M/E ratio: 3:1, 3. Myeloid series 4. Erythroid series 5. Lymphocytes:
7. Plasma cells: are characterized by eccentric nucleus, paranuclear hallow, basophilic
cytoplasm.
8. Myeloma cells: Multinucleated in the form of bi, tri, quadric nucleated forms, loss of
perinuclear hallow.
9. Blasts 9. Megakaryocytes 10. No Haemoparasite
11.No other abnormal or malignant infiltration at examined site.
Conclusion: BM is markedly hypercellular, infiltrated by 90% cells of plasmacytic series → a
case of MM for: total protein, SPE (monoclonal band) – bone x-ray, CT → for osteolytic
lesion.

Case 4: A case of Megaloblastic anemia:

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 Clinical Hematology

1.BM Cellularity & M/E ratio: markedly hypercellular with ↓ or reversed M/E ratio 1:2
3.Myeloid series: Hypersegmented, neutrophils, giant metamyelocyte & giant band forms.
4.Erythroid series: marked Erythroid hyperplasia showing megaloblastic changes.
-Stippled appearance of nuclear chromatin.
-Nuclear cytoplasmic asynchrony: nuclear maturation lags behind that of cytoplasm.
-Howel jolly bodies - Mitotic figures.
8. Megakaryocytes: are mildly increased both juvenile and mature forms with adequate
platelets separation.

Case 6: A case of Iron deficiency anemia:

1. BM Cellularity: Markedly hypercellular with Erythroid hyperplasia ↓
M/E ratio (or reversed). Predominance of late normoblast, delayed Hbinization with ragged
cytoplasm , micro-Normoblastic reaction.

Case 7: A case of megakaryocytes (ITP or hypersplenism).

a. ITP:
- No Organomegaly
- ↓ platelets + ↑ megakaryocytes + no platelets separation
- Decrease or absent platelets around megakaryocyte
- Decrease pseudopodia of megakaryocytes (no evidence of platelets budding)
a. Hypersplenism: as ITP but with organomegaly and late
erythroid.

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 Clinical Hematology

Case 8: Aplastic anemia

- In PB: pancytopenia + Macrocytosis + ↓ Retics + ↓ RPI.

- No LNs or organomegaly.
- At BM: markedly hypocellular (> 75% fat) with relative ↑ in lymphocytes, plasma cells.
Patchy hypocellular BM.
- Other investigation:
 ↑ ESR, NAP score
 Trephine biopsy: replacement of marrow by fat patchy cellular areas in a hypocellular
background, decrease reticulin fibres.

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NOTES
1. If Auer rods are present: AML
2. M3: hypergranular promyelocytic leukemia + pancytopenia (blast + pro ≥ 20%) + DIC
3. L3: Burkitt’s cell type → vacuolation in blasts in nucleus and cytoplasm.
4. M6: acute erythroleukemia (PAS)
Important Notes:
1. Pathogenic of leukemia is due to dysregulation of:
- Balance between proto-oncogenes and tumor suppressor genes
- -ve apoptosis
- Cell cycle check points (P53, P16, P17).
2. Investigation: CBC – BM – ESR – Retics – IPT – Cytogenetic.
3. CML diagnosed by: ↓ NAP score or zero – cytogenetic t (9:22) (BCR/ABL).
4. AML markers → CD13, CD33, CD117.
5. ALL markers → B-ALL → CD19,20,23 T-ALL → CD 2,3,5,7
6. Lymphoma → CD22, 79bt, FMC7
7. M6 → glycophorin A, CD13,33

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