Manual Testing in Hematology

Use of Peripheral Blood Smear

Used to analyze blood component microscopically - often created when an automated instrument flags an issue

How many identifiers should be on the PBS

At least 2 - name and accession number

When are albumin smears made for PBS

When there is increased number of smudge cells. Only a WBC diff is done on this smear.

How to prevent water artifact on a PBS with low hemoglobin

Let them dry longer

Characteristics of a good peripheral blood smear

• Smooth even film with a feathered end

• Minimum of 1 inch length

• Blood should not carry off the edge or spill over the side

What causes a PBS to be too short

Blood spread too quickly, angle of spreading is >30 degrees

What causes a PBS to be too long

Spreading blood too slowly, angle of spreader <30 degrees

What causes a PBS to be too thin

Too little blood, low Hgb

What causes a PBS to be too thick

Drop of blood too large, patient abnormalities with viscous blood

Pre-test sample requirement for malaria testing

Travel history and indication

How many smears are required in the malaria protocol

8: 4 thin smears, 4 thick smears

What is the use of thick and thin smears for malaria

Thin smears: regular PBS to calculate parasitemia

• 2 are stained and examined in routine heme labs, 2 are forwarded to special hem for %parasitemia

Thick smears: used to examine for presence of malaria

• All 4 are sent right to special heme

How to label malaria slides

Normal indicators, and include MAL or MALARIA or some indication that slides are for malarial examination

Purpose of creating buffy coat smears

Analyze and evaluate blood smears for Bacteria/parasites that may have been observed/questioned on the PBS. Diffs and RBC is not performed

If a PBS shows bacteria and the buffy coat shows extracellular bacteria, what does this indicate?

That there is bacterial contamination on the slides, the stainer, etc.

If a PBS shows bacteria and the buffy coat shows intracellular bacteria, what does this indicate?

Patient is septic

Winthrope tube method for buffy coat (steps)

1. Label 2 wintrobe tubes with patient name and accession number

2. Fill the wintrobe tube with EDTA whole blood and parafilm the top (approx. 0.8mm)

3. Insert wintrobe tube into test tube, secure with gauze

4. Centrifuge for 1500g for 10 minutes

5. Remove excess plasma (leave some above buffy coat, approx. 2mm)

6. Sift off the buffy coat and remaining plasma into separate tube (labelled microtube)

7. Repeat for 2nd tube, combine patient into one tube to increase sample yield

8. Prepare 2 smears and label according to protocol + label with buffy coat

9. Stain the slides with routine hematology staim

When is a cytospin differential indicated?

When the WBC count is above the reference range in order to aid in determining the increased WBC count in CSF or fluids

What is the use of a cytocentrifuge?

Concentrates fluids which normally have relatively low concentrations of cells

How does a cytoscentrifuge work

Slides are placed in centrifuge and the patient sample is added while soinning. Low speed of the centrifuge minimizes cell distortion and concentrates them into a button

How to look under a microscope for fluid analysis

Entire cell button scanned under 10X to search for large abnormal cells or tumor cells

50X or 100X used for cell differentiation

Any area of the cell button can be used for the differential

Use of bone marrow analysis

Determine overall cellularity and quantitate cells present in the bone marrow. Can also be stained differently to look for elements such as iron, reticulinm etc.

What is used instead of a BM aspirate if the bone marrow is hypocellular or there is increased fat/reticulin

Trephine or hole punch biopsy is used and processed by histo. These samples allow for evaluation of bone marrow

BM sample type for cell morph and diff

1mL aspirate in EDTA

Sample type of BM aspirate for immunophenotyping

1mL aspirate in bone marrow media at 4C

Sample type of trephine biopsy

2.0cm sample in a sterile collection container with gauze

What happens if there is no particles present in BM aspiration (no solid marrow tissue in the aspirate)

Another aspiration may need to be performed. Granules/Particles must be obtained for megakaryocyte analysis

BM Push smear

BM smear that can be used for differentials. One drop of aspirate on a glass slide pushed to make a ‘smear’. This is stained using routine hematology stains and special stains if requested. 8 push smears should be made.

BM Squash smear

used for megakaryocyte analysis. A BM particle is placed on a glass slide, and a second slide is used to squish the particle between the glass slides

BM touch prep biopsy smear

Trephine biopsy is lightly rolled and touched between two slides. Prepared in heme before fixation/processing for histo

Contnets of the Wright GIemsa Stain

Methylene blue, purified azure B, eosin.

• Also requires ethanol and aged DH2O

• pH 6.8

Use of each component of the Wright Giemsa Stain

Methanol - fixes cells and enhances dye uptake

Methylene blue and azure B (basic dyes) - stain acidic components: RNA/DNA, nucleic acids
Eosin - acidic dye stains basic components of cells; hemoglobin, eosinophil granules

Neutral pH of dye allows for staining of neutrophil granules

DH2O acts as buffer to improve contrast of cellular materials

Contents of the May-Grunwald-Giemsa Stain

May Grunwold contains eosin and methylene blue

Giemsa contains: eosin, purified azure B, methylene blue

Requires phosphate buffer and water at pH 6.8

Use of each stain component in May Grunwald Giemsa

MG - primarily cytoplasmic (does not demonstrate nuclear detail, PTLs, or malaria inclusions)

Giemsa - nuclear stain demonstration inclusions such as malaria, but does not stain RBC or neutrophil granules well

Common supravital stains and their uses

New methylene blue (retics, Heinz bodies)

Brilliant Cresyl Blue (retics, Heinz bodies)

Methyl violet, Crystal violet, Brilliant green = Heinz bodies

Contents of Perl’s priussian blue stain

0.2N HCl, Potassium Ferrocyanide. Also requires methanol and 0.2% aqueous safranin

Perl’s prussian blue staining principle

Slides are fixed in methanol

● Slides are incubated in the HCL and potassium ferrocyanide mixture.

● The HCL splits the ferric iron from hemosiderin

● Ferric ions combine with potassium ferricyanide to form a blue complex

● Only ferric irons are demonstrated – ferritin cannot be demonstrated as it is too small to

be seen with light microscopy)

● Slides are counterstained with aqueous safranin to increase contrast

Use of Perl’s Prussian Blue Stain in Heme

Demonstrates iron stores in BM Particle smears. Results are reported as decreased, normal, or increased.

What conditions cause increased iron stores in BM

Megaloblastic anemia

Hemolytic anemia

Sideroblastic anemia

Anemia of Chronic Disease

Lead Poisoning

Hemosiderosis

What conditions cause decreased iron stores in BM

IDA

Polycythemia vera

Use of MPO Stain

Stains primary granules in monocytes and myeloid lineage

Use of SBB stain

Stains primary and secondary myeloid granule lipids, weakly stains granules in monocytes

Use of PAS stain in Heme

Stains glycogen compounds found in lymphoid lineage

Use of NSE stain

Stains non-specific esterase compounds found in monocytes

Use of Leukocyte Alkaline Phosphatase staining

Can differentiate leukemoid reactions from CML, as leukemoid reaction shows increased staining

Contents and requrements of LAP stain

Contains: naphthol phosphate, Fast red violet salt, fast blue salt

Requires: Acetone, hematoxylin solution for counterstain

What conditions cause increased LAP staining

LAP can be increased in:

Leukemoid reaction

Multiple myeloma

Hodgkin’s disease

Myeloproliferative disorders such as Polycythemia vera (except CML)

Aplastic anemia

What conditions can cause decreased/minimal LAP staining

Chronic myelogenous leukemia (CML)

Paroxysmal nocturnal hemoglobinuria

Sickle cell anemia

Myelodysplastic syndromes

Effect of high pH on Hemeatology staining

• increased dissociation of methylene blue = stain is too blue

• RBC stain green/blue

• Neutrophils appear toxic

Effect of too low pH on Hematology staining

Increased dissociation of eosin causing the stain to be very pink

• RBC stain red-orange

• WBC nuclei will be pale

• Eosin granules are increasingly bright orange

What casues stain precipitate

Inadequate washing, stain drying on slide precipitated stain powder in solution requiring filtration

Effect of water artifact on hematology staining

Causes halos on RBCs (cells appear hypochromic and refractile)

When is a WBC correction performed

When >/= 2 nRBCs are present per 100 WBC in the blood smear

Corrected WBC formula

Automated WBC count / (100+nRBCs) x 100

Use of Miller Ocular Lens in Hemetology

Used for retic counts and malarial parasitemia levels.

Are gametocytes counted as infected RBCs for malaria

No. They are extracellular

How to calculate %parasitemia/%reticulocytes

%parisitemia = (Square B x 100) / Square A x 9)

What can be included in a laboratory evaluation of body fluids

Total volume, gross appearance, total cell count, differential cell count, crystal IDs, biochemical analysis, microbial examination, immunological studies, cytological examination

What evaluation of body fluids do hematology labs perform

• Gross examination [appearance (color, clarity) - spun and unspun]

• WBC count (TNC) - RBCs only reported if requested

• Cytospin differential (if number of TNCs is >200)

What fluids are grouped together and known as serous flouids

CSF, Pleural, Peritoneal, Pericardial Fluids

What are serous fluids

Ultrafiltrates of plasma found in small amounts of the pleural, peritoneal, and pericardial cavities, where they serve as lubricant.

What is a transudate

An accumulation of body fluid caused by a non-inflammatory circulator disturbance. There will be an increase or deacrease in pressure that results in fluid formation

Examples of disorders that result in transudates

• CHF (heart can’t pump properly = fluid backup and leakage)

• Liver disease (decrease in albumin = decrease in oncotic pressure)

• Renal disease (loss of albumin = decrease in oncotic pressure)

• Obstructive tumors (poor lymph drainage)

What is an exudate

Excess amount of fluid caused by an inflammatory condition such as infection, malignancy, SLE or rheumatoid arthritis. There is an accumulation of fluid in association with vascular wall damage

What is CSF

An ultrafiltrate of plasma across the blood-brain barrier. Examining CSF is important in conditions such as meningitis, brain hemorrhage, or CNS involvement by leukemia)

What hematology testing is done on CSF

Gross examination of color/cllarity

Cell counts (TNC and RBC)

Cytospin differential (if number of WBC indicates)

What CSF tube is the hematology cell count performed on

The last one, as it has the least amount of cells present from the patient collection

When would a physiican order a CSF cell count on the first tube

When they want to rule our brain hemorrhage - must do cell counts on first and last tibe

Are CSFs older than 2 hours since recieved still worked up?

Yes, but a comment is made about the results.

What happens if there is any macroscopic abnormality in CSF (color or clarity)

A part of sample must be spun down for the spun appearance to determine if xanthochromia is present (orange/yellow color indicating bilirubin from cerebral hemorrhage)

What causes cloudy and colorless bodily fluids

High WBCs, microorganisms

Does the laboratory ever label incorrectly labeled fluid tubes?

No, but we allow the ordering ward to send a witness to label the samples before analysis occurs

Use of ordering crystal identification on synovial fluid

To ID joint fluid for uric acid

Clinical significance of increased reticulocytes in peripheral blood

Increase in RBC production or accelrated erythropoiesis. May be due to:

• Hemorrhage

• Hemolysis

• Hypoxia

• Hematinic injury

When must a manual reticulocyte count be done

If the automated reticulocyte count is:

• Present with an interference flag

• Relative result >30%

• Absolute result >90 × 10^9

Use of ESR test (erythrocyte sedimentation rate)

Non-specifc test used as a marker for tissue damage or inflammation. Can be increased by infection, RA, TB, Cancer

Why does ESR increase in multiple myeloma?

In the presence of excess proteins (like immunoglobulins), Zeta potential is decreased, allowing for RBCs to form rouleaux, stack, and fall quicker than normal

Tube requirement for ESR

EDTA

How is ESR testing performed

Blood is aspirated into a ESR westergren tibe and allowed to rest for one hour. Once the RBCs are settled, the distance from the botom of the plasma meniscus to the sedimented RBC is recorded in mm

Effect of abnormally shaped cells on ESR

Decreased (slower settling)

Effect of increased number of RBCs/WBCs on ESR (leukemias, PV)

Decreased (more crowding, thcieker blood)

Effect of anemia on ESR

Increased (less crowding, thinner blood)

Effect of microcytosis and macrocytosis on ESR

Microcytosis: Decreased (light and smaller cells fall slowly)

Macrocytosis: Increased (heavier cells fall quickly)

General effect of Plasma proteins on ESR

Plasma proteins counteract negative charge of RBCs (as they are positive) and increase possibility of Rouleaux formation. Therefore increased levels of fibrinogen, immunoglobulin, and albumin all decrease ESR

Mechanical factors that effect ESR testing

Tilted tube increases ESR (shorter distance to travel)

Cold temps decrease ESR (increased viscovity)

Warm temps increase ESR

Excess EDTA decreases ESR (EDTA causes RBCs to shrink)

Delay in setting up test decreases ESR (shape changes)

Vibration from centrifuge increases ESR

Use of Monospot testing

Red cell or latex agglutination tests that detect heterophile antibodies that the body produces in response to infectious mononucleosis

How does the BioSign Mono Immunoassay test work?

Bovine RBCs are used in a direct solid-phase immunoassay. If heterophile Abs are present in patient blood, they will bind and be captured by an antigen bad (made out of bovine RBCs).

After, a conjugated dye is mobilized to provide visual indication on the presence or absence of the antibodies and a QC line

What does the osmotic fragility test demonstrate

Increased RBC fragility in specimens in which RBCs have decreased SA to Vol ratios (seen in Hereditary Spherocytosis)

How does Osmotic fragility testing work

RBCs are subjected to increasingly hypotonic solutions, so water rushes into the RBCs to equilibrate. Cells with a smaller SA to Vol ratio will lyse more easily, and cells with a larger ratio (target cells) will take up more water without lysing.

How can confirmatory testing for G6PD deficiency be done?

A G6PD kinematic enzymatic assay

How does the G6PD kinematic enzyme assay work?

Patient RBCs are lysed, and the released G6PD will convert glucose-6-phosphate into 6-phosphogluconate, which reduces NADP to NADPH proportionally. The rate of production is measured as the absorbance at 340 nm by a spec

What is a quick screening test for G6PD?

Same kinematic enzymatic G6PD reaction, but the fluorescence of NADPH is measured

How can Pyruvate Kinase levels be tested for PK deficiency?

Quantitative Enzymatic Assay

How does the PK Enzymatic assay work

Patient RBCs are lysed and WBCs removed (as WBC’s falsely increase results). Released PK converts phosphoenolpyruvate into pyruvic acid while converting ADP into ATP.

The pyruvic acid reacts with LDH to be converted into Lactic acid, while NAD is converted into NADH. The rate of converseion of NAD to NADH is measured at 340 nm with a spec

How can a screening test be done for PK deficiency

Same enzymatic test method as the quantitative test, but NADH fluorescence is used to screen

Effect of reticulocytes + transfusion on patient PK levels

Sources of interference when performing a PK assay. Must be noted before performing.