blood and urine.docx

### Fibrin strands - Poor coagulation - Incorrect distribution of cells internally ### Reference for Cell Counting: Before the Feathery Edge **First Part:** Too thick; RBCs are clumped together **Before Feathery Edge Part:** - Cells are at ideal distances - More visible and easier to...

### Fibrin strands - Poor coagulation - Incorrect distribution of cells internally ### Reference for Cell Counting: Before the Feathery Edge **First Part:** Too thick; RBCs are clumped together **Before Feathery Edge Part:** - Cells are at ideal distances - More visible and easier to count **Feathery Edge Part:** - Cells are distorted - Not suitable for counting ### staining Crystal Violet (Gram Staining) and Blood: - Used for smear staining - Alternative Staining Procedure - Used when no methanol is available - Methanol is a fixative that makes cells stick to the slide and kills the cells Supravital Staining: 1. Colors cells while they are still alive 2. Used to observe, count, and identify reticulocytes (immature RBCs seen in peripheral blood smear) High reticulocyte count indicates a higher rate of RBC production Reticulocytes are normally seen under a microscope Common Stains for Reticulocytes: 1. New methylene blue 2. Crystal violet 3. Brilliant cresyl blue ### Characteristics of Reticulocytes 1. Larger than normal RBCs 2. Have dots (lines - reticulum fiber/networks) inside the cytoplasm Lipid residue from fingerprints on slides can appear as bubble-like dots ### reason for high reticulocyte count - Anemia due to red blood cells being destroyed earlier than normal ([hemolytic anemia](https://www.mountsinai.org/health-library/diseases-conditions/hemolytic-anemia)) - Bleeding (including menstruation) - Blood disorder in a fetus or newborn (erythroblastosis fetalis) - Kidney cysts or tumors, with increased production of a hormone called [erythropoietin](https://www.mountsinai.org/health-library/tests/erythropoietin-test) - Pregnancy ### reason for low reticulocyte count - Anemia caused by low iron levels, or low levels of vitamin B12 or folate - Bone marrow failure (for example, from a certain drug, tumor, radiation therapy, or infection) - [Chronic kidney disease](https://www.mountsinai.org/health-library/diseases-conditions/chronic-kidney-disease), due to decreased amounts of a key hormone - [Cirrhosis of the liver](https://www.mountsinai.org/health-library/diseases-conditions/cirrhosis) ### Cell Measurements and Values 1. 11.5 - 14.5: Normal red cell width (size comparison among cells) 2. Normal RBC size: 6.5 μm 3. Mean Corpuscular Volume (MCV): 80-100 (normal range) 4. MCV, Mean Corpuscular Hemoglobin Concentration (MCHC), and Mean Corpuscular Hemoglobin (MCH) are used for anemia classification 5. Central pallor of RBCs: Should be 1/3 of the total diameter (normal) urine strip ----------- 1. Leukocyte - 120 seconds 2. PPUBN -- 60 seconds - Protein - pH - Urobilinogen - Nitrite 3. Specific Gravity -- 45 seconds 4. Ketone -- 40 seconds 5. GLUBIL -- 30 seconds Ascorbic acid at higher concentration in urine samples can lead to **false negative results** in several urine tests, with a potential risk of clinical findings being overlooked, particularly with glucose and hemoglobin. principles ---------- **Glucose: Double Sequential Enzyme Reaction -- Green to Brown** - **Principle:** The test for glucose in a sample typically involves two sequential enzyme reactions. First, glucose oxidase catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide. In the second step, peroxidase catalyzes the reaction between hydrogen peroxide and a chromogen, producing a color change from green to brown. The intensity of the color change is proportional to the concentration of glucose present in the sample. **Bilirubin: Diazo Reaction -- Tan or Pink to Violet** - **Principle:** The diazo reaction involves the coupling of bilirubin with a diazonium salt to form a colored compound. When bilirubin in a sample reacts with a diazo reagent, it produces a tan, pink, or violet color. The presence and intensity of the color indicate the concentration of bilirubin, which is significant in diagnosing liver function and hemolytic diseases. **Ketone: Sodium Nitroprusside Reaction -- Purple** - **Principle:** This reaction detects acetoacetic acid (a type of ketone) in urine. When acetoacetic acid reacts with sodium nitroprusside under alkaline conditions, it produces a purple color. This test is commonly used to monitor ketosis in diabetes and other metabolic conditions. **Specific Gravity: pKa Change of Polyelectrolytes -- Blue** - **Principle:** The test measures the specific gravity of a urine sample by detecting changes in the pKa of polyelectrolytes. These polyelectrolytes release or bind hydrogen ions in response to the ionic concentration of the urine, causing a color change. A blue color typically indicates higher specific gravity, reflecting higher solute concentration. **Protein: Protein Error of Indicator -- Blue Green** - **Principle:** This test is based on the protein error of certain pH indicators. In the presence of protein, indicators such as tetrabromophenol blue change color without a change in pH. The protein in the sample binds to the dye, resulting in a blue-green color change. This test is often used to detect proteinuria, a sign of kidney disease. **pH: Double Indicator System -- Orange** - **Principle:** A double indicator system uses two pH indicators to provide a broad pH range. The test pad contains two different indicators that produce a color change based on the pH of the urine. An orange color typically represents a neutral pH. **Blood: Pseudoperoxidase Activity of Hemoglobin -- Dot or Speckles** - **Principle:** The presence of blood in urine is detected by the pseudoperoxidase activity of hemoglobin. This activity catalyzes the oxidation of a chromogen by hydrogen peroxide, resulting in a color change that appears as dots or speckles on the test pad. This test can indicate hematuria or hemoglobinuria. **Urobilinogen: Ehrlich Reaction -- Red** - **Principle:** Urobilinogen in a sample reacts with Ehrlich\'s reagent (p-dimethylaminobenzaldehyde) to produce a red color. This reaction is used to assess liver function and hemolysis, as increased urobilinogen levels may indicate liver disease or hemolytic disorders. **Nitrite: Greiss Reaction -- Uniform Reaction** - **Principle:** The Greiss reaction detects nitrite, which is produced by the reduction of nitrate by bacteria in urine. In the presence of an acidic environment, nitrite reacts with aromatic amines to form a diazonium compound, which couples with another compound to produce a pink color. A uniform color change indicates the presence of nitrite, suggesting a urinary tract infection. **Leukocytes: Esterase / Hydrolysis Reaction -- Purple** - **Principle:** Leukocyte esterase, an enzyme present in white blood cells, hydrolyzes an ester on the test pad, producing an aromatic compound that reacts with a diazonium salt to form a purple color. This reaction indicates the presence of leukocytes, which may suggest an infection or inflammation in the urinary tract. automation and hematological analyses **introduction** ---------------- Automation is machine-based analysis of hematological samples where the following are detected: 1. Cell count 2. Cell population (nuclear and cytoplasmic granulations) Subpopulations of cell (flow cytometry) 3. Cell counting and reading (dating nagagawa ng machines) **T Cell Subpopulations:** Machines can differentiate between the three types of lymphocytes (T cells, B cells, and Natural Killer cells) using flow cytometry. By employing specific fluorescent markers that bind to surface antigens on these cells, the analyzers can distinguish between: - **T Cells:** Identified by markers such as CD3, with subtypes like CD4+ (helper T cells) and CD8+ (cytotoxic T cells). - **B Cells:** Identified by markers like CD19 or CD20. - **Natural Killer (NK) Cells:** Identified by markers such as CD16 and CD56. ### electronic impedance Measures changes in electrical resistance as cells pass through a small aperture 1. LVDC (Low-Voltage Direct Current) 2. RF (Radio Frequency) - Alternating Current - Used in conjunction with Direct Current ### optical scatter Use laser light to detect and count cells based on their size and granularity. This technique is often employed in flow cytometry. 1. Laser 2. Non-laser **electronic impedance** ------------------------ ### terminologies to consider 1. Oscilloscope : volume distribution histogram - Y-axis = relative number - X-axis = volume 2. Proprietary lytic/diluting reagents - 3-part differentials 3. Aperture diameter - RBC / Platelet - Granulocyte - Mononuclear cells 4. Sample Stream ### measurements 1. Platelets : 2 -- 20 femtoliters 2. Erythrocytes : \>36 femtoliters 3. Lymphocytes : 35 -- 90 femtoliters 4. Mononuclear cells : 90 -- 160 femtoliters 5. Granulocytes : 160 -- 450 femtoliters ### errors in instrumentation **Burn circuits / washings** - maintenance feature to prevent protein buildup - Protein buildup - occur if multiple samples are run through the analyzer without proper cleaning. When proteins accumulate in the apertures, it can obstruct cell passage, leading to inaccurate cell counts. - Cells Carryover - when a sample is processed, cells may remain in the system and carry over into the next sample. This carryover can contaminate the results of the following patient sample, leading to inaccurate cell counts. **Aperture passage** refers to the small opening through which cells pass during analysis. Several factors can alter the passage of cells through the aperture: - **Orientation of Cells:** The position or orientation of cells as they pass through the aperture can affect the readings. Cells may align differently, impacting the measurements. - **RBC Deformability:** The ability of red blood cells (RBCs) to change shape and squeeze through the aperture can influence their passage. Stiff or poorly deformable RBCs may affect the accuracy of cell counts and size measurements. **Coincident passage** occurs when two or more cells enter the aperture simultaneously and are counted as a single cell. This leads to inaccuracies in cell counts, known as **coincident passage loss.** - Correction: Modern hematology analyzers use mathematical algorithms to correct for coincident passage by estimating and adjusting the counts based on statistical models. **Recirculation** refers to the process where blood cells that have passed through the analysis tube are returned to the external part of the system instead of being disposed of immediately. - Purpose: Recirculation can help maintain a steady flow of cells for consistent analysis and can also be used to reanalyze cells to verify results. However, proper system design ensures that recirculated cells do not contaminate subsequent samples. ### improvements in instrumentation 1. Rigid aperture system 2. Hydrodynamic focusing - aperture has fluid sheath to: - allow laminar flow - minimizes protein buildup - eliminate recirculation - reduce pulse heigh irregularities **radio frequency** ------------------- 1. High-voltage electromagnetic current - interacts with cells, providing information about their physical and chemical properties. 2. Measures the attenuated conductivity of: - Nucleus : Cytoplasm -- nuclear density and cytoplasmic granules 3. Used in conjunction with the Direct Current Impedance ; detected simultaneously - DC impedance measures cell volume based on changes in electrical resistance as cells pass through an aperture. 4. **FIVE PART DIFFERENTIALS** - distinguishing the five main types of white blood cells: neutrophils, lymphocytes, monocytes, eosinophils, and basophils. **Sources of error** -------------------- ### instrumental error 1. Aperture plugs are probably the most common problem in cell counting **(positive)** - cell gain 2. Extraneous electric pulses from improperly grounded or shielded equipment may be picked up by the instrument electrode **(positive)** 3. Improper setting of aperture current or threshold (**positive or negative**) 4. Bubbles in the sample caused by too vigorous mixing (**positive**) 5. Excessive lysing of RBC (**negative**) due to alcohol and water application ### errors caused by specimen nature 1. **Giant platelets** can be mistakenly counted as red blood cells (RBCs) or white blood cells (WBCs) 2. **Fragments of leukocyte cytoplasm**, such as may be present during leukemia therapy, may be counted as platelets or RBCs, which can result in falsely elevated RBC or platelet counts. 3. An **increased number of schistocytes**, which are fragmented RBCs, may be counted as platelets because of their small size, causing falsely elevated platelet counts and potentially masking thrombocytopenia. 4. **Agglutination of RBCs, WBCs, or platelets** results in false-negative cell counts, as these clumps may be mistakenly counted as a single large cell, typically a WBC, when passing through the analyzer. 5. **Platelet satellitism**, where platelets adhere to WBCs, causes a falsely low platelet count because the analyzer may not count them as separate entities; if suspected, a blood smear is performed, and a sample is redrawn using a citrate tube to reduce platelet adhesion and provide more accurate counts. 6. **Agglutinated red cells or platelets** can be miscounted as WBCs due to their increased size when clumped, leading to falsely elevated WBC counts and inaccurate RBC and platelet counts. 7. **Some abnormal RBCs tend to resist lysis**, which may result in high WBC counts. Examples are SICKLE CELLS, EXTREMELY HYPOCHROMIC CELLS, and TARGET CELLS. The problem can be solved by delaying 2-3 minutes between the addition of the lysing agent and counting. ### important 1. **Normal RBCs in Histogram:** In a normal RBC histogram, the peak should be near but below 100 fL, indicating the typical size range of red blood cells. 2. **Small RBCs:** If the RBCs are small, the peak will be positioned far from 100 fL on the histogram, indicating microcytic cells. 3. **Large RBCs:** When RBCs are large, the peak exceeds 100 fL, suggesting macrocytic cells. 4. **Variation in RBC Sizes:** A variation in RBC sizes is reflected in the red cell distribution width (RDW), which shows the degree of size variability. 5. **Platelets on Histogram:** Platelets typically appear in the size range between 2 and 20 fL on the histogram. 6. **Leukocyte Peaks:** Leukocyte histograms typically show three distinct peaks: - **Lymphocytes:** The first peak appears between 35-90 fL, corresponding to lymphocytes. - **Mononuclear Cells:** The second peak, ranging from 90-160 fL, includes monocytes, blasts, immature granulocytes, and reactive lymphocytes. - **Granulocytes:** The third peak occurs between 160-450 fL, representing mature granulocytes. **optical scatter** ------------------- 1. Used by FLOW CYTOMETER systems 2. Hydrodynamic focusing is present 3. Light sources - Tungsten-Halogen Lamp (Non-laser) - Helium-Neon (Laser) 4. Laser is better because of its: - Intensity - Coherence -- single unified string - Low spread ### optical scatter principle 1. Light scatter when interrupted by a passing cell in the sensing zone 2. Components: Lenses with block bars - captures the scattered light then passes it to the photodetector 3. Photodetector : - Photodiode - Photomultiplier tube - placed at 90-degree detection. Capable of amplifying the amounts of lights captured and very sensitive than photodiode. ### terminologies to consider 1. Forward-angle light scatter 2. Orthogonal light scatter (side scatter) - Absorption - Retraction - Reflection - Diffraction 3. Forward-low angle scatter (2-3 degrees) 4. Forward-high angle scatter (5-15 degrees) - Differential scatter in the combination of these dual angle scatter used is SIEMENS Technology. NOTE: The different angular scatter detection is the basis of plotting the differential scatter plot as in ABBOT CELL-DYN Technology additional inputs ----------------- ### expanded base of wbc 1. Not testing the sample in recommended time 2. Medication esp. chemotherapy drugs ### others 1. Macrocytic RBC - vitamin b12 and folic acid deficiency 2. Microcytic - hemoglobin deficiency ### ABNORMALITIES 1. 3rd peak is high = neutrophilia (increased number of neutrophils) 2. 1st peak is high = lymphocytosis (increased number of lymphocytes) ### RBC DISTRIBUTION WIDTH - differences of rbc sizes 1. Anisocytosis - variability in sizes 2. Anisochromia - variability in color **Mean Cell Hemoglobin** - average weight of hemoglobin expressed in picograms **Mean Cell Volume** - average volume of rbc expressed in femtoliters.