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Summary

Perform a differential on a blood slide, comment on red cells, white cells and platelets, make a diagnosis and suggest follow up confirmatory tests using an Aiforia link.Calculate indices and suggest possible causes of these result Counts and Indices  Red blood cell counts o Red blood cell count (r...

Description

Perform a differential on a blood slide, comment on red cells, white cells and platelets, make a diagnosis and suggest follow up confirmatory tests using an Aiforia link.

Calculate indices and suggest possible causes of these result Counts and Indices 

Red blood cell counts o Red blood cell count (rbcc) - number of cells/volume o Haemoglobin concentration (hb) – weight hb/volume o Haematocrit (hct) - % of rbc/volume o Red cell distribution width (RDW) - variation in cell size • Reticuloctye count cells/volume or % of rbc

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Red cell indices (calculated) o Mean cell volume (MCV) (hct/rbcc) – volume of each rbc o Mean cell haemoglobin (MCH) (hb/rbcc) - amount of haemoglobin in each rbc o Mean cell haemoglobin concentration (MCHC) (hb/hct) – concentration of haemoglobin in each rbc

Counts and indices SI (conventional) 12

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Red blood cell count (rbcc) ~ 4-6 x 10 /L

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Haemoglobin concentration (hb) ~ 150 g/L (15g/dL) • Haematocrit (hct) ~ 0.45 (45%)

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Mean cell volume (MCV) (hct/rbcc) ~ 85 fL

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Mean cell haemoglobin (MCH) (hb/rbcc) ~ 30 pg

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Mean cell haemoglobin concentration (MCHC) (hb/hct) ~ 330 g/L (30g/dL)

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Red cell distribution width (RDW) ~ 11-15 (A measure of variation in cell size/volume)

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Reticulocyte count ~ 50-150 x 109/L (0.2-2%)

Interpretation of Indices 

MCV – Mean Cell Volume (Size of the cells) 80 – 100 fL, fL = 10-15L o Increased MCV – means your cells are BIG (> 100fL) You would then describe them as being MACROCYTIC Causes: Fe Deficiency, Thalassemia (will have a high RBC) o Normal MCV – means your cells are the NORMAL (85fL) You would then describe them as being NORMOCYTIC Causes: Blood loss, Haemolytic anaemia o Decreased MCV – means your cells are SMALL (< 80fL) You would then describe them as being MICROCYTIC Causes: B12 and Folate Deficiency, Liver Disease

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MCH – Mean Cell Haemoglobin (Amount of hb in each cell) 27 – 32 pg/cel o Increased MCH – means your cells have too MUCH (> 32 pg/cel) You would then describe them as being HYPERCHROMIC (dark appearance) o Normal MCH – means your cells have a NORMAL amount (30 pg/cel) You would then describe them as being NORMOCHROMIC (normal appearance) o Decreased MCH – means your cells have too LESS (< 27 pg/cel) You would then describe them as being HYPOCHROMIC (light/pale appearance)

RBC < Lymphocyte (size) Iron deficiency anaemia

Hypochromic and Microcytic

Normochromic macrocytic Megaloblastic anaemia

Hyper-segmented Neutrophils

Anaemia 

Reduced O2 delivery to tissues – low or defective Hb

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Signs /symptoms: Pallor, lethargy, poor exercise tolerance, dyspnoea, faintness, nausea

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Causes: increased blood loss & RBC destruction, decreased RBC production

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Classification: o Hypochromic / microcytic, E.g., Iron deficiency, thalassaemia o Normochromic / normocytic, E.g., Acute blood loss o Normochromic / macrocytic, E.g., Megaloblastic anaemia, Vitamin B12 &/or folate deficiency

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Diagnosis: Hb, Hct, Rbcc, film o MCV, MCH, MCHC o RDW o ± bone marrow examination o ± specific assays

Nor malRanges 

Red cell count (Rbcc) o 5.0 ± 0.5 x1012/L (adult male) o 4.3 ± 0.5 x1012/L (adult female)

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Haemoglobin (Hb) o 150 ± 2.0 g/L (adult male) o 135 ± 1.5 g/L (adult female)

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Haematocrit (Hct) (Percentage red cells in sample)

o 45 ± 5 % (male) (0.45) o 41±5 % (female) (0.41) 

Normal variations o Age, gender, altitude

RedCel lDi sor der s Nor mal er y t hr ocy t emor phol ogy -Bi conc av eDi s cs ,7um andcent r alpal l or Needatl eas t10% v ar i abi l i t ybef or ewec al li tabnor mal

Poikilocytosis – Variation in Shape Cell Elliptocytes

Description Long and thin Shape only when mature Not Significant

Spherocytes

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Perfectly round and dense. No area of central pallor Significant -> Pathological Disorder

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Target Cell

Target cells can be seen in several diseases, using MCV can help you decide which disease you are looking at.

-

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Significant Schistocyte

A very significant finding. Sharp pointy edges, wedge or pizza shaped. Very Significant even 1% is significant, imminent death if not reported

Oval Macrocyte

They look like fat juicy elliptocytes. Has High MCV

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Example of Disorders Hereditary elliptocytosis (Membrane defect) 20 –70% elliptocytes Iron deficiency Thalassaemia ≤10% elliptocytes

Hereditary spherocytosis Membrane defect (works but body thinks it’s bad so gets rid of it) Autoimmune haemolytic anaemia (splenic macrophages gets rid of it) Burns (Micro-spherocytes) (splenic macrophages remove bad parts)

Liver Disease: Increase MCV (Attacks outside membrane forcing it to collapse on itself), excess membrane Thalassaemia, Fe Def. (microcytic, hypochromic, Decreased MCV (RBC lost structural integrity, empty, therefore collapses on itself) Trauma in circulation, defective cardiac valves, essentially holes in the heart Haemolytic anaemia, microangiopathic, small clots form in capillaries. When other RBC’s come they get torn about.

B12 and folate deficiencies -> Make RBC bigger Seen in conjunction with hyper-segmented neutrophils

Have at least 5 lobes

Round Macrocyte

Large round cell still has some central pallor.

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Liver Disease with target cells Can be Dysplasia Seen with some drugs and medication

High MCV

Tear drop cell

Myelofibrosis: formed when the red cell is squeezed out of the bone marrow. Fibrotic marrow is hard not spongey therefore RBC’s comes out squeezed.

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Thalassaemia

Burr cell

Plasma Factors

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Dehydration Uraemia Renal Failure

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Seen post splenectomy and in end stage liver disease.

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Crenation when blood has sat in EDTA for too long Oxidative haemolysis (only time you see it) G6PD deficiency

Only significant in population

Acanthocyte/ spur cell

Spherocyte with pointy projections Lacks Central Pallor Variation in membrane lipids

Bite/Blister Cell

NOT Crenation -> Artifact Looks like someone took a bite out of the edge of the cell. Significant Spleen eats it to kill it

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Sickle cell

Dense cells with no central pallor, pointy ends -

Abnormal Hb HbS HbSC Get stuck in circulation as they are rigid (lack flexibility)

HbS–Ami noac i ds ubs t i t ut i oni nbet agl obi nc hai n Pr obl em

RBC Inclusions Cell Rouleaux

Description Stacking off red blood cells in chains on the blood film. -

Make sure correct area Howell-Jolly body

Pappenheimer Body

Nuclear or Chromosomal Remnants Looks like Santa Clauses belly RBC -Nuclear Biconcave disc Iron Particle: Seen with an iron stain

Example of Disorders Is seen in inflammation or when there is excess paraprotein Will also have a high CRP and ESR

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Seen post splenectomy

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Indication: Abnormal ion synthesis

Body is precipitated iron

Basophilic Stippling

Altered Hb Synthesis

-

Pathological condition

Heinz Bodies

Unstable Hb (Precipitated, denaturated Hb)

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Significant – Oxidated homolysis

Appear as bite cells in normal film NB Supravital stain eg. Methyl violet, Cresyl blue

Erythropoiesis – From blue to red Cell Pro-erythroblast

Description -

12 – 20 um High Nuclear: Cytoplasmic Ratio Nucleoli Present Basophilic Cytoplasm (Blue)

Basophilic Erythroblast

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10 – 16 um Reduced Nuclear: Cytoplasmic Ratio Nucleoli Absent (Difference) Condensed Nuclear Chromatin Basophilic Cytoplasm

Polychromatic erythroblast

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8 – 14 um Reduced Nuclear: Cytoplasmic Ratio Checkerboard clumping of nuclear chromatin Increased Hb (hence cytoplasm polychromatic) Patchy Nucleus Purple & White

Orthochromatic erythroblast

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8 – 10 um Small, pyknotic nucleus Increased Hb If seen there is an abnormality

Reticulocytes

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8 – 9 um Anucleate Polychromatic Cytoplasm Only seen with supra-vital stain If on blood film -> Polychromasia RBC bigger, bluer

Mature erythrocyte

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7 um Eosinophilic Cytoplasm RBC’s

Anisocytosis – Variation in Size Described as + to +++ directly related to red cell width (RDW) Description Example of Disorders = 9um, Big Determined by MCV

Hypochromic

Very Pale Low conc. in haemoglobin Determined by MCH

Hyperchromic

2 Cell populations dimorphic->ANISOCYTOSIS Dark High conc. In haemoglobin Determined by MCH

Normal

Central Pallor 7 um Can be seen in MCH & MCV

Examine films and comment on their correlation to indices

Discuss safe work laboratory practices

Read and interpret simple tests such as ESR/MONOSPOT/COAGULATION and other manual haematology methods and discuss their importance and limitations.

ESR 

Erythrocyte Sedimentation Rate (ESR), A test to measure the rate at which red cell settle within 1 hr.

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Independent of the red cell count or Hct. Units are in mm/hr.

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Increases in females over males due to different hormones.

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Increased in inflammation and in some malignant conditions where there is a high paraprotein such as multiple myeloma.

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Associated with the patients density of plasma

Video: How to read an ESR (Links to an external site.)

Manual Laboratory Tests Manual Tests

Heinz Bodies

Indicators



Exposure to

Procedure

Clinical Significance

Spra vital stain

Sign of chemical poisoning,drug toxicity, G6PD deficiency,

oxidising agents (bite cells) 

Unstable Hb

Wet prep

unstable Hb

Dry films

Sickle Cell

• HbS

Reduction agent +

HbSS or compound

Test

• Sickle cell anaemia

blood

heterozygote, HbAS

sealed wet prep Rbcs deprived of O2

G6PD screen

Fluorescent screening

Drug induced HA

spot test- (G6PD

Haemolytic crisis in infection or

deficient patients will

diabetic acidosis, Favism

not fluoresce)

neonatal hyperbilirubinemia

Fluorescent screening

Chronic nonspherocytic HA,

Kinase

spot test, enzyme

PKU(Phenylketonurics- genetic

screen

assay

brain disease)

Cytospin urine deposit

Intravascular haemolysis-

perform iron stain

haemosiderinuria, PNH

ESR tubes

Infection or haem disorder eg.

Pyruvate

Urinary

Haemolysis

Haemolysis

Haemolysis

Haemosideri n

ESRs

Inflammation/ infection

BM Aspirate

Haem Malignancy

MM

BM aspirates

& Fe

trephines special

Staining

staining

Malaria

Overseas travel

Staining

lethargy rigors

ICT kit test

Leukaemias MPD, MDS

Plasmodium falciparum, vivax,

fevers

Malarial thin and thick

cwcplt

films

malariae, ovale, knowlesi

MP staining

Monospot

Swollen glands

Monospot kit, blood

lethargy

films

Infectious monoucleosis

lymphocytes

Kleihauer

Mother Rh neg mum

Elutions/ staining films

APH/PPH

Prophylactic Anti D, fetal haemorghage

MONOSPOT Monospot test 

The monospot test is a rapid latex kit for screening for infectious mononucleosis

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It tests for the presence of heterophile antibodies (antibodies against other

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species)

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It will generally not be positive during the 4–6-week incubation period before the onset of symptoms

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It will also not generally be positive after active infection has subsided, even though the virus persists in the same cells in the body for the rest of the carrier's life

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Commercially available test kits are 70-92% sensitive and 96-100% specific

Monospot Test Method 

A Kit may contain o Latex beads (e.g. coated with Paul Bunnell antigen in buffer) o Card with different sections for each test o Positive control (e.g. Rabbit antibody against Paul Bunnell antigen in buffer) o Negative control (non-reactive diluted human serum or buffer only) o Patient sample for testing: Plasma or serum

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Steps 1. Allow reagents to warm up to room temperature 2. Gently shake the latex reagent to resuspend latex particles in buffer 3. Add one drop each of patient serum, pos control and neg control to 3 different sections on the card 4. Place a drop of latex beads on each of the 3 sections 5. Mix all the drops with a stirrer (change stirrers for each sample)

6. Gently rotate slide for 3 min 7. Look for the presence or absence of agglutination 

Heterophile antibody (in patients’ serum due to exposure to EBV, reacts to RBC from other species) + horse RBC (in kit) = agglutination = positive



No antibody + horse RBC = no agglutination = negative

https://www.youtube.com/watch?v=qb_4sT7RvZc

Coagulation Screen 

Prothrombin time (PT)

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Activated partial thromboplastin time (APTT)

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Thrombin time (TT)

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Uses platelet poor plasma (PPP) (Just to look at coag. factors)

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Reagents to initiate clotting

Prothrombin Time (PT) 

Screening test for abnormalities of ‘extrinsic pathway’ o Factors II, VII, X and I



PT measures clotting of plasma in presence of tissue factor (thromboplastin) and Ca+ + o Normal: 12-16 seconds

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Prolonged PT o FVII deficiency o Liver disease

o Warfarin

International Normalised Ratio (INR) 

PT results vary based on tissue factor reagent used

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INR used to monitor patients on warfarin or related oral anticoagulant therapy o The normal range for a healthy person 0.8–1.2 o For people on warfarin therapy an INR of 2.0–3.0 is usually targeted

Activated partial thromboplastin time (APTT) 

Screening test for abnormalities of ‘intrinsic pathway’ – Factors other than FVII



APTT measures clotting time of plasma with contact activator (e.g., kaolin), a platelet substitute (phospholipid) and Ca++

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Normal: 25-35 seconds

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Prolonged APTT – FVIII or FIX deficiency

Thrombin Time (TT) 

Screening test for ‘common pathway’

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Measure’s conversion of fibrinogen to fibrin

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TT requires plasma and thrombin (containing Ca++) o Normal: 13-17 seconds

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Prolonged TT o Fibrinogen deficiency

Specific Coagulation Assays 

Fibrinogen assay



Specific assays for FV, FVIII, FIX

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Assays for other coagulation factors

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Screen assays so we can first narrow it down (cost & time efficient)

Case Study I A 5-yr-old male child presented with history of spontaneous swelling of right calf for 2 days, recurrent joint swelling, past 2 years, and prolonged bleeding in elder brother. Examination revealed that he had mild pallor, diffuse swelling of right calf, contracture of left knee joint with marked restriction of joint movements Investigations 

Hb: 120g/L ↓

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Platelet count: 160 x109/L N

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Bleeding Time: 3 min N

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PT: 15 sec N

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APTT: 90 sec ↑

Most likely diagnosis= Haemophilia A; confirmatory tests → FVIII levels Case Study II A 3-yr-old female child presented with petechial haemorrhages and repeated nosebleeds. The patient had a history of easy bruising, repeated gum bleeding, but not hemarthrosis. There was no family history of abnormal bleeding Investigations: 

Hb: 58g/L ↓

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Platelet count: 100 x109/L ↓

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Bleeding Time: 15 min ↑

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PT: 14 sec N

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APTT: 30 sec N

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Platelet aggregation tests: ADP: 5% ↓ ↓

Most likely diagnosis = Platelet function disorder (Glanzmann thrombasthenia) Confirmatory tests: flow cytometry, genetic analysis

Read and interpret haematology analyser print outs and scatterplots. Interpret error codes and discuss steps that can be taken to rectify the error. Discuss limitation of automation.

Interference can be seen when the curve does not start and end on the x -axis e.g., look at the circles Histograms 

Dimorphic Population Histograms All 4 are

not normal: (↑ RDW) A) MCV=94fl RDW= ↑18.1% Normocytic with minor macrocytic component

B) MCV=112fl RDW= ↑24.1% Most volume High proportion of macrocytes

C) MCV= 65fl RDW= ↑29.2% Normocytic with a continuum of small red cells &/ fragmented cells

D) MCV=75fl RDW= ↑26.7% Bimodal or DIMORPHIC RBC DISTRIBUTION. 25%

Mixture of microcytic and normocytic rcc Seen after blood transfusion&/treatment of microcytic anaemia

Automation Gone Bad 

Automation is not perfect, there are several occasions where interference occurs, and incorrect results are obtained:

1. Cold agglutination 2. Spherocytosis 3. Lipaemia 4. Abnormal platelets 5. Abnormal differentials Cold Agglutination

Spherocytosis

Lipaemia &/Haemolysis

Platelets

Abnormal WCC differential: DON’T IGNORE THE FLAGS

AIFORA slides https://cloud.aiforia.com/Public/UTS_Gorrie29092020/9xPOYQERN3vdgGCIazbVkNKNe1m2L8oTeAYKLyloyw0#!/slidepageid=MQPYJDkMAHqfUhmEtN37YvtvI8e19Enstfb2p2a TNik0§ionid=86881e73-6b56-4698-bd2f-9d119dc1f672 Hyper segmented Neutrophils

https://cloud.aiforia.com/Public/UTS_Gorrie29092020/ZDMqeEW1ox14lVBiw6qtJEFb84ZQDXJcb5a4gFtMI0#!/slidepageid=MQPYJDkMAHqfUhmEtN37YvtvI8e19Enstfb2p2aTNik0§ionid= 7f301d2e-adb6-4050-a74e-3f1ebef39d37 Toxic granulation

https://cloud.aiforia.com/Public/UTS_Gorrie29092020/GqY6pvOPEhpCKFf1A1Jk3SGxYK 4ErfvqdflebXRmQfY0#!/slidep...