Hematology Safety and Isolation Techniques

Enhanced Hematology: Safety and Quality

• Standard precautions apply to blood, semen, vaginal secretions, CSF, pleural fluid, visible blood fluids, unidentified body fluids, unfixed slides, microhematocrit clay, and saliva from dental procedures.
• A material safety data sheet describes hazards and safe handling, storage, and disposal of hazardous wastes.
• OSHA mandates that laboratories maintain a personal protective equipment program.
• The sequence for donning PPE is gown, mask or respirator, goggles or face shield, then gloves.
• The sequence for doffing PPE is gloves, goggles or face shield, gown, mask or respirator, then handwashing.

Isolation Techniques

• Strict/Complete Isolation: Used for contagious diseases spread by direct contact via air, PPE includes gown, mask, and gloves.
• Enteric Isolation: Used when contacting patients with dysentery/related disorders spread by direct contact, PPE includes gown and gloves.
• Respiratory Isolation: Used for infections transmitted by droplets or airborne routes, PPE includes gown, mask, and gloves.
• Wound and Skin Isolation: Used for potentially directly/indirectly transmitted skin infections.
• Protective/Reverse Isolation: Protects patients (e.g., leukemia, burn, transplant patients) from infections, PPE includes gown, mask, and gloves.

Quality and Standards

• ISO 15189:2007: Used to develop quality management, assess competence, and for accreditation of medical laboratories.
• Quality assessment ensures excellence in hematology lab performance, with non-analytical factors and quantitative data analysis (QC).
• Non-analytical quality assessment factors include qualified personnel, lab policies, procedure manuals, test requisitioning, and proper patient/specimen ID and labeling.
• Pre-analytical error examples: wrong patient, wrong time, wrong tube, wrong order, incorrect labeling, improper processing.
• Analytical error examples: oversight of instrument flags, out-of-control QC results, wrong assay.
• Post-analytical error examples: verbal reporting, LIS incompatibility, confusion about reference ranges, failure to report critical values.
• Specimen collection, transport, processing, storage, equipment maintenance, methodology, and reporting affect quality.
• Quality control in hematology monitors test accuracy/precision over time to detect errors from system failure, adverse conditions, or variance.

Blood Specimen Collection

• Venipuncture tourniquets compress veins and restrict blood flow; apply 3-4 inches above site for ≤1 minute.
• Needles include multisample, hypodermic, winged infusion, and nonwinged blood collection types. Needle gauge: 19-23, Length: 1.0-1.5 inches. Adult venipuncture common size: 21 gauge, 1 inch, Angle: 15-30°.

Order of Draw

• Sterile Yellow SPS/Sterile Media Bottles, use: minimizes microbial contamination.
• Light Blue: Coagulation tubes, use: this affects coagulation tests.
• Red: Glass tubes (non-additive), use: prevents contamination by additives. Filled after coagulation tests as silica affects clotting.
• SSTs: Red and gray rubber Gold plastic, use: Serum-separator tubes.
• PSTs: Heparin tubes (Green and gray rubber Light-green plastic) use: Heparin affects coagulation tests/interferes with serum but is least interfering other than that.
• EDTA (Lavender, pink, purple Pearl/white top) use: Responsible for carryover problems; elevates Na+/K+, chelates Ca2+/Fe, elevates PT/PTT.
• Gray: Oxalate/fluoride tubes, use: Affect Na+/K+ (collect after hematology tubes).

Venipuncture Complications

• Complications include ecchymosis, hematoma, fainting, hemoconcentration, hemolysis, petechiae, allergies, nerve damage, seizures, and vomiting.

Venipuncture Special Situations

• Special situations include edema, obesity, burned/damaged/scarred/occluded veins, IV therapy, and mastectomy patients.
• Skin puncture is preferred for newborns/pediatric patients.

Manual Blood Smear

Standard Procedure:

• Drops of blood should be 2-3 mm/0.05mL in size.
• Held in 30-45° angle on the slide.
• Characteristics of an ideal blood smear include a tick to thin gradient, covers 2/3-3/4 of the slide length, finger shaped, with visible borders.

Potential Issues:

• RBCs appearing gray or blue indicates stain/buffer is too basic/inadequate mixing/heparinized blood.
• Dark WBCs - can mean overstaining.
• Reddish tinting - too acidic.
• Hard-to-see WBCs - underbuffering.

Manual Cell Count:

• Most common is Levy chamber with improved Neubauer ruling.

General Formula

• cells counted x dilution factor / area (mm²) × depth (0.1). Alternatively cells counted X dilution factor X 10 / area (mm²).

Manual Cell Counts Common Amounts

• White Blood Cells: 1:20/1:100 using 1% ammonium oxalate/3% acetic acid/hydrochloric acid and 10X objective for measuring cell count over 4/9mm².
• Red Blood Cells: 1:100 dilution using Isotonic saline at 40X objective to count blood cell over 0.2mm².
• Platelets: 1:100 - use of 1% ammonium oxalate at 40x phase to count cell over 1mm².

Erroneous Blood Smear

• Grayish/blue RBCs or overly dark WBCs (too basic).
• Pale/reddish RBCs or barely visible WBCs (too acidic).

Factors Determining Thin or Thick Film

• Thin Film: ↑ pressure, ↓angle, ↓specimen size, and ↓speed.
• Thick Film: ↑specimen size and ↑speed; opposite of thin.

Testing Methods

• Tubes for blood gas analysis come first in collection for skin puncture.
• Slides tested prior to EDTA in microcollection.
• Serum tubes at the tail end of sampling.

Hemoglobin Determination

• Uses Drabkin solution that consists of potassium ferricyanide, potassium cyanide, sodium bicarbonate, and a surfactant Absorbance is determined at 540nm.
• Can measure all forms except sulfhemoglobin.
• Sources of error include:
  • High WBC/platelet count.
  • Lipemia
  • Presence of Hb S or C
  • Anomalous globulins

Measuring Hematocrit

• Is the measure of volume packed RBC occupies in a whole-blood portion.
  • Uses microhematocrit tube of appx. 74mm in length and a .05mL capacity.
  • Blue-band/red-band types.
  • Rule of Three states X3 Hg Concentration = Hematocrit (+/-3%) and is used for verification purposes regarding normocytic analysis.
  • Only works in normocytic/chromic specimens.

Errors:

• False increase - dehydration, hemoconcentration, incomplete centrifuge, buffy coat.
• False decrease - hemolysis, incomplete sealing, anti-coagulation.

Cell Indices

• MCV - mean cell volume, the RBC's average volume, expressed in femtoliters HCT (%) × 10 / RBC count (×10¹²/L)
• MCH - mean cell Hemoglobin, weight of the hemoglobin, results in pictograms g/dL) × 10 / RBC count (×10¹²/L)
• MCHC - concentration of hemoglobin. HGB (g/dL) × 100 / HCT (%)

Erthyrocyte Sedimentation Rate

• Measures to detect inflammation. Has a direct association with aggregate weight but is inversely proportional to plasma of viscosity

Reticulocyte Count

• Shorter-than-normal erythrocyte survival is flagged with this test, indicator of rate of erythrocyte production.
• Reticulocytes % = # of Ret. X 100 / 1000 X RBCs.

Miller-Disc

• Designed to make RBC %: # reticulocytes in square A (large square) × 100/# RBCs in square B (small square) × 9

Corrected Reticulocyte Count

• Is the early release of what appears to be great numbers of erythrocyte as a result of limited production.
• Is measured as the percentage of Ret. X (PT HCT%) / 45

Reticulocyte Production Index

• Percentage calculation of Ret. that factors for the prematurely released Ret. that need 0.5-1.5 more days to mature
• Ret. (%) X (Hct%/45) / Maturation time.

Potential normal manual reading variants:

• RBC - is 10-20%
• WBC - is 15%
• Ret - is +/-25

Cell Morphology and Associated Disorders

Red Blood Cells

• Anisocytosis: cells come in varying volume/diameter - implies a number of anemias.
• Macrocytic: oversized in anemic patients and those w/ liver disease.
• Microcytic: undersized for iron deficiencies.
• Poikilocytosis: distorted RBC in all types of anemia.
• Spherocyte: No central impression means hemolytic or microspherocytic.
• Elliptocyte: Elliptical shapes mean iron deficiency
• Stomatocyte: Slit-like impression means kidney disease
• Schistocyte: Fragments/ruptured cells w/micro-hemolytic expression.
• HbC Crystal: Shaped crystalline structures tied congenital defects.
• Hb S Expression: Elongated with pointed ends due to sickle cell.
• Target morphology- Thalassemia.
• Burr: Small, spread w/evenly distrubuted projections indicative of uremia.
• Teardrop variants: Tear-shaped as result of myelofibrosis (marrow scarring).
• Acanthocyte: Projectiles of an irregular shape result from severe liver defect.
• Basophillic traits: Punctured granulites result following a number of deficiencies.
• Howell-Jolly bodies: After one lacks a spleen and there remnants of removed DNA in the bloodstream.

Hemolysis

• Extravascular - RBC's destruction outside the boundaries of blood being tested as well as macrophages causing disruption, or kidney, or other tissues.
• Intravascular destruction causes breaking of blood cell; hemoglobin spills out of the tissues.

Anemia

• Results in decreased quantity of hemoglobin.
• Causes can include blood loss, impeded blood cell generation, destruction by increased pace

Iron Deficiency Anemia

• Results in: intake of iron that is limited to the standard level of demand, need of it expanding but intake doesn't catch-up. Lack of absorption, or chronic blood loss with hemoglobin.

Microcytic and Macrocytic Differentiation

• MCV <80fL - Microcytic group includes sideroblastic and Thallassemic based expressions.
• HVC > 100fL indicates Macro based variances (B12 deficiencies and liver disease).

White Blood Cells

• Leucocytosis measures white blood cell creation and the development.

• All processes occur in bone narrow/lymph nodes.

• Myeloblast: has a 4:1 Ratio.

• Promyelocytes: Cytoplasm ratio (3:1 to 2:1), Pale blue-basophillic color.

• Myelocyte: Ratio of 4:1 Chromatin that has compacted cytoplasm with few blue patches.

• Metamyelocte Ratio of 1:1- specific granules are seen.

• Band:

• Nucli Elongated, indented Cytoplasm alike Meta.

• Cytoplsam Same meta.